Constitutive activation of the Saccharomyces cerevislae mating response pathway by a MAP kinase kinase from Candida albicans

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.Communicated by C. P. Hollenberg     

Flavonol glycosides from Chenopodium foliosum Asch

Three new flavonol glycosides, namely 6-methoxykaempferol-3-O-β-gentiobioside, gomphrenol-3-O-β-gentiobioside and gomphrenol-3-O-α-l-rhamnopyranosyl-(1 → 2)[β-d-glucopyranosyl-(1 → 6)]-β-d-glucopyranoside as well as the known patuletin-3-O-β-gentiobioside and spinacetin-3-O-β-gentiobioside were isolated from the aerial parts of Chenopodium foliosum Asch. The structures of the compounds were determined by means of spectroscopic methods (1D and 2D NMR, UV, IR, and HRMS). DPPH free radical scavenging activity of the new compounds was low or lacking.     

Effects of triptolide from Tripterygium wilfordii on ERα and p53 expression in two human breast cancer cell lines

The aim of the study was to discover possible differential cytotoxicity of triptolide towards estrogen-sensitive MCF-7 versus estrogen-insensitive MDA-MB-231 human breast cancer cells. Considering that MCF-7 cells express functional Estrogen receptor α (ERα) and wild-type p53, whereas MDA-MB-231 cells which are ERα-negative express mutant p53, the anti-proliferation effect of triptolide on MCF-7 and MDA-MB-231 cells were examined, the apoptotic effect and cell cycle arrest caused by triptolide were investigated, ERα and p53 expression were also observed in this paper. The results showed that the anti-proliferation effects were induced by triptolide in both cell lines. But the value of IC₅₀ in MCF-7 cells for its anti-proliferation effect was about one tenth of that in MDA-MB-231 cells, which indicated that the effect is more potent in MCF-7 cells. Condensed chromatin or fragmented nuclei could be found in MCF-7 cells treated with only 40 nM triptolide but in MDA-MB-231 cells they couldn’t be observed until the concentration reached to 400 nM. Triptolide induced significant S cell cycle arrest along with the presence of sub-G0/G1 peak in MDA-MB-231 cells, whereas there was only slightly S cell cycle arrest on cell cycle distribution in MCF-7 cells. The role of p53 in two breast cancer cells was examined, the results showed that the mutant p53 in MDA-MB-231 cells was suppressed and the wild-type p53 in MCF-7 was increased. Moreover, triptolide could down regulate the expression of ERα in MCF-7 cells. The results showed that triptolide is much more sensitive to ERα-positive MCF-7 cells than to ERα-negative MDA-MB-231 cells, and the sensitivity is significantly associated with the ERα and p53 status.     

Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

Growth inhibitory activity of extracts and compounds from Cimicifuga species on human breast cancer cells

The purpose of this report is to explore the growth inhibitory effect of extracts and compounds from black cohosh and related Cimicifuga species on human breast cancer cells and to determine the nature of the active components. Black cohosh fractions enriched for triterpene glycosides and purified components from black cohosh and related Asian species were tested for growth inhibition of the ER⁻ Her2 overexpressing human breast cancer cell line MDA-MB-453. Growth inhibitory activity was assayed using the Coulter Counter, MTT and colony formation assays.Results suggested that the growth inhibitory activity of black cohosh extracts appears to be related to their triterpene glycoside composition. The most potent Cimicifuga component tested was 25-acetyl-7,8-didehydrocimigenol 3-O-β-d-xylopyranoside, which has an acetyl group at position C-25. It had an IC₅₀ of 3.2 μg/ml (5 μM) compared to 7.2 μg/ml (12.1 μM) for the parent compound 7,8-didehydrocimigenol 3-O-β-d-xylopyranoside. Thus, the acetyl group at position C-25 enhances growth inhibitory activity.The purified triterpene glycoside actein (β-d-xylopyranoside), with an IC₅₀ equal to 5.7 μg/ml (8.4 μM), exhibited activity comparable to cimigenol 3-O-β-d-xyloside. MCF7 (ER⁺Her2 low) cells transfected for Her2 are more sensitive than the parental MCF7 cells to the growth inhibitory effects of actein from black cohosh, indicating that Her2 plays a role in the action of actein. The effect of actein on Her2 overexpressing MDA-MB-453 and MCF7 (ER⁺Her2 low) human breast cancer cells was examined by fluorescent microscopy. Treatment with actein altered the distribution of actin filaments and induced apoptosis in these cells.These findings, coupled with our previous evidence that treatment with the triterpene glycoside actein induced a stress response and apoptosis in human breast cancer cells, suggest that compounds from Cimicifuga species may be useful in the prevention and treatment of human breast cancer.     

Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.     

In vitro cytogenetic effects of Andrographis paniculata (kalmegh) on arsenic

In vitro effects of arsenic in human peripheral lymphocytes (HPL) at three different doses – 3.6×10⁻⁴, 1.4×10⁻³ and 0.72×10⁻³ μM for 24 h before harvesting on sister chromatid exchanges (SCE), Cell cycle proliferative index/replicative index (CCPI/RI), %M₁, %M₂ and %M₃, population doubling time (PDT) and average generation time (AGT) were examined. Andrographis paniculata (commonly referred to as ‘kalmegh’) has been used for centuries in traditional Indian and Chinese herbal medicine as a safe, natural folk remedy for assorted health concerns. In the present study, kalmegh (0.01 μg/7 ml culture media) was used along with the highest dose of arsenic; the results showed that arsenic induced increase in these genotoxic endpoints were fairly diminished by kalmegh. In addition, mutagenic in vitro effect of ethyl methanesulphonate (EMS) was used as a positive control in this study. It is thus concluded from this study that Andrographis has a protective role in arsenic toxicity.     

Acylated flavonol diglucosides from Lotus polyphyllos

Three acylated flavonol diglucosides, kaempferol 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; quercetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; isorhamnetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside were isolated from the whole plant aqueous alcohol extract of Lotus polyphyllos. The known 3,7-di-O-glucosides of the aglycones kaempferol, quercetin and isorhamnetin were also characterized. All structures were established on the basis of chemical and spectral evidence.     

爵床的甲醇提取物对小菜蛾的生物活性作用

为探明爵床(Justicia procumbens)甲醇提取物对小菜蛾的生物活性,采用室内生测法测定了爵床甲醇提取物对小菜蛾的触杀、拒食、胃毒、生长发育抑制和产卵忌避作用。结果表明,爵床甲醇提取物对小菜蛾幼虫具有较强的触杀、拒食、胃毒和生长发育抑制活性,对小菜蛾成虫具有较强的产卵忌避活性。在触杀试验中,药后1、2 d和3 d爵床甲醇提取物对小菜蛾3龄幼虫的致死中浓度(LC50)分别为5.17、4.05和3.06 mg/m L;在拒食试验中,药后1 d和2 d提取物对3龄幼虫的选择性拒食中浓度(AFC50)分别为2.64和3.13 mg/m L,药后1 d和2 d提取物对3龄幼虫的非选择性拒食中浓度(AFC50)分别为3.70、4.54 mg/m L;在胃毒试验中,药后4、5、6 d和7 d提取物对3龄幼虫的致死中浓度(LC50)分别为8.13、3.65、2.88、2.23 mg/m L;在生长发育抑制试验中,药后1 d和2 d提取物对3龄幼虫的抑制中浓度(IC50)分别为2.02、1.40 mg/m L;在产卵忌避试验中,药后1、2 d和3 d提取物对小菜蛾成虫的选择性产卵忌避中浓度(AOC50)分别为2.61、3.66、4.58 mg/m L,药后1、2和3 d提取物对小菜蛾成虫的非选择性产卵忌避中浓度(AOC50)分别为3.19、4.52、5.65 mg/m L。由此证实,爵床提取物对小菜蛾具有显著的毒杀活性,具有开发为新型高效、低毒植物源农药的潜在价值。     

Anti-tumor potential of ethanol extract of Curcuma phaeocaulis Valeton against breast cancer cells

Curcuma phaeocaulis Valeton is a commonly prescribed Chinese medical herb for tumor therapy. In this study, an extract of Curcuma phaeocaulis Valeton referred as Cpv was prepared and its anti-tumor effect was evaluated with MCF-7 and MDA-MB-231 cells. Curcuma phaeocaulis Valeton power was extracted with ethanol and the main components of the extract (Cpv) were analyzed with HPLC. The effect of Cpv on MCF-7 cells proliferation, intracellular reactive oxygen species (ROS) formation, mitochondrial membrane potential (ΔΨm), apoptosis, apoptotic related proteins, MDA-MB-231 cell migration, and integrins expression were determined. Furthermore, the effect of Cpv on some key signal transduction molecules was also investigated. Furanodienone, germacrone and furanodiene were identified as the main components of Cpv. Cpv treatment significantly inhibited cell proliferation, increased LDH release, induced intracellular ROS formation, and decreased ΔΨm in a dose-dependent manner in MCF-7 cells. Cpv induced apoptosis without affecting cell migration. Cpv increased protein expression of Bax, PARP, cleaved PARP, caspase-3, 7, JNK1, p-p42/44MAPK, NF-κB, IKKα, IKKβ, decreased protein expression of Bcl-2, Bcl-xL, Bim, Bik, Bad, integrin β5, p42/44MAPK without affecting integrin α5, β1, and p38MAPK protein expression. We concluded that Cpv inhibited MCF-7 cells proliferation by inducing apoptosis mediated by increasing ROS formation, decreasing ΔΨm, regulating Bcl-2 family proteins expression, and activating caspases. Cpv treatment also modulated several signaling transduction pathways. These results might provide some molecular basis for the anti-tumor activity of Curcuma phaeocaulis Valeton.     

铁皮石斛内生真菌次生代谢产物研究

为了解铁皮石斛(Dendrobium officinale)内生真菌Phyllosticta aristolochiicola的次生代谢产物,从该真菌中分离得到15个化合物,经波谱分析分别鉴定为N-methyl-2-pyrolidinone (1)、环-(甘氨酸-L-脯氨酸)(2)、环-(D-丙氨酸-L-脯氨酸)(3)、环-(L-缬氨酸-L-脯氨酸)(4)、环-(L-亮氨酸-L-脯氨酸)(5)、cyclo-(L-Leu-D-4-hydroxyprolinyl)(6)、环-(L-苯丙氨酸-L-脯氨酸)(7)、环-(L-苯丙氨酸-L-4-羟基脯氨酸)(8)、环-(L-酪氨酸-L-脯氨酸)(9)、环-(L-苯丙氨酸-L-亮氨酸)(10)、啤酒甾醇(11)、对羟基苯乙醇(12)、对羟基苯乙酸(13)、(2S,3R)-1-(4-羟基苯基)丁烷-2,3-二醇(14)和(2R,3S)-1-苯基丁烷-2,3-二醇(15)。采用MTS法检测抗肿瘤活性表明,化合物2、10和14对HL-60、A-549、SMMC-7721、MCF-7和SW-480细胞株具有一定的抑制活性。     

Characterization of an Azospirillum brasilense Sp7 gene homologous to Alcaligenes eutropbus pbbB and to Rbizobium meliloti nodG

Summary A 4 kb SalI fragment from Azospirillum brasilense Sp7 that shares homology with a 6.8 kb EcoRI fragment carrying nodGEFH and part of nodP of Rhizobium meliloti 41 was cloned in pUC18 to yield pAB503. The nucleotide sequence of a 2 kb SalI-SmaI fragment of the pAB503 insert revealed an open reading frame, named ORF3, encoding a polypeptide sharing 40% identity with R. mehloti NodG. The deduced polypeptide also shared 60% identity with the Alcaligenes eutrophus NADPH-dependent acetoacetyl-CoA (AA-CoA) reductase, encoded by the pbbB gene and involved in poly--hydroxybutyrate (PHB) synthesis. Northern blot analysis and promoter extension mapping indicated that ORF3 is expressed as a monocistronic operon from a promoter that resembles the Escherichia coli ⁷⁰ consensus promoter. An ORF3-lacZ translational fusion was constructed and was very poorly expressed in E. coli, but was functional and constitutively expressed in Azospirillum. Tn5-Mob insertions in ORF3 did not affect growth, nitrogen fixation, PHB synthesis or NAD(P)H-linked AA-CoA reductase activity. An ORF3 DNA sequence was used to probe total DNA of several Azospirillum strains. No ORF3 homologues were found in A. irakense, A. amazonense, A. halopraeferens or in several A. lipoferum strains.     

In vitro estrogenic activity of Achillea millefolium L.

Isolation and biological characterization of pure compounds was used to identify and characterize estrogenic activity and estrogen receptors (ER) preference in chemical components of Achillea millefolium. This medicinal plant is used in folk medicine as an emmenagogue. In vitro assay, based on recombinant MCF-7 cells, showed estrogenic activity in a crude extract of the aerial parts of A. millefolium. After fractionation of the crude extract with increasing polar solvents, estrogenic activity was found in the methanol/water fraction. Nine compounds were isolated and characterized by HR-MS spectra and 1D- and 2D-NMR techniques. In particular, dihydrodehydrodiconiferyl alcohol 9-O-beta-D-glucopyranoside - a glycosyl-neolignan - was isolated for the first time from the genus Achillea in addition to six flavone derivatives, apigenin, apigenin-7-O-beta-D-glucopyranoside, luteolin, luteolin-7-O-beta-D-glucopyranoside, luteolin-4'-O-beta-D-glucopyranoside, rutin, and two caffeic acid derivatives, 3,5-dicaffeoylquinic acid and chlorogenic acid. Apigenin and luteolin, the most important estrogenic compounds among those tested, were studied for their ability to activate alpha or beta estrogen receptors (ERalpha, ERbeta) using transiently transfected cells. Our results suggest that isolation and biological characterization of estrogenic compounds in traditionally used medicinal plants could be a first step in better assessing further (e.g. in vivo) tests of nutraceutical and pharmacological strategies based on phytoestrogens.     

Fumigant toxicity of the essential oils of some African plants against Anopheles gambiae sensu stricto

The essential oils from 15 species of African plants selected by ethnobotanical considerations and field inspection (odour and presence of insects) were screened for fumigant toxicity to Anopheles gambiae s.s. in the laboratory. Essential oils from 6 species showed varying levels of toxicity, with Conyza newii (Compositae) and Plectranthus marruboides (Labiateae) being the most potent. Fifty compounds representing approximately 74% of the essential oil of C. newii were identified by GC-MS and GC-coinjection (for available standards). The major and some of the minor constituents of the two oils were assayed at different doses. Two compounds, from C. newii, perillaldehyde and perillyl alcohol, exhibited higher fumigant toxicity (LD50 = 1.05 x 10(-4) and 2.52 x 10(-4) mg cm(-3), respectively) than the parent oil (2.0 x 10(-3) mg cm(-3)). GC-MS analysis of the essential oil of P. marruboides gave results similar to that previously reported. Interestingly, none of its components were active, suggesting that the insecticidal activity of the oil results from either some of the minor components or as a blend effect of some of the major constituents.     

白花除虫菊化学成分及其生物活性的研究

该研究应用柱层析法、薄层层析法和波谱法对白花除虫菊(Pyrethrum cinerariifolium)全株甲醇提取物进行分离纯化、结构鉴定,并分别采用 MTT 法、生物活性测定法和抑菌圈法测定所得化合物抗肿瘤、杀线虫和抑菌活性。结果表明：经波谱法鉴定化合物结构分别为 tulirinol (1),sesamin (2),β-cyclopyrethrosin (3)和3,5-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-7,8-dimethoxy-4H-1-benzopyran-4-one (4)。抗肿瘤活性显示化合物3对白血病细胞株 HL-60、肝癌细胞株 SMMC-7721、肺癌细胞株 A-549、乳腺癌细胞株 MCF-7和结肠癌细胞株SW480均表现出显著的抑制活性,其 IC50分别为3.800、2.890、2.930、4.600和5.160μmol?L-1;化合物1活性比3稍弱,其 IC50分别为5.020、10.760、12.310、12.310和12.250μmol?L-1;其中化合物3对各肿瘤细胞株 IC50值均小于顺铂。抗菌活性表明化合物3对大肠杆菌、蜡样芽孢杆菌、枯草芽孢杆菌和金黄色葡萄球菌均表现出明显的抑菌活性,且随着浓度增加活性逐渐增强,而化合物1和化合物2仅对部分菌株表现出微弱抑菌活性;杀线虫活性显示化合物3对秀丽隐杆线虫和全齿复活线虫均表现出显著的毒杀活性,且随着时间、浓度增加活性逐渐增强;而在同一时间点对秀丽隐杆线虫的毒杀活性强于全齿复活线虫。从白花除虫菊中分离所得4个化合物均为首次从该植株中发现,且首次报道了化合物3杀线虫和抑菌活性,值得进一步开发应用。     

Neural cell protective compounds isolated from Phoenix hanceana var. formosana

A platform for screening drugs for their ability to protect neuronal cells against cytotoxicity was developed. Nerve growth factor (NGF) differentiates PC12 cells into nerves, and these differentiated PC12 cells enter apoptosis when challenged with 6-hydroxydopamine (6-OHDA). A screening spectrophotometer was used to assay cytotoxicity in these cells; pretreatment with test samples allowed identification of compounds that protected against this neuronal cytotoxicity. The 95% ethanol extract of Phoenix hanceana Naudin var. formosana Beccari. (PH) showed potential neuroprotective activity in these assays. The PH ethanol extract was further fractionated by sequential partitioning with n-hexane, ethyl acetate (EtOAc), n-butanol (n-BuOH), and water. Subsequent rounds of assaying resulted in the isolation of ten constituents, and their structures were characterized by various spectroscopic techniques and identified by comparison with previous data as: isoorientin (1), isovitexin (2), veronicastroside (3), luteolin-7-O-β-d-glucopyranoside (4), isoquercitrin (5), tricin-7-neohesperidoside (6), tricin-7-O-β-d-gluco-pyranoside (7), (+)-catechin (8), (−)-epicatechin (9), and orientin 7-O-β-d-glucopyranoside (10). Among these compounds, isovitexin (2), luteolin-7-O-β-d-glucopyranoside (4) and (+)-catechin (8) showed significant neuroprotective activity in cell viability (WST-8 reduction), anti-apoptosis (Annexin V-FITC/propidium iodide double-labeled flow cytometry), and cellular ROS scavenging assays (besides isovitexin (2)), as well as a decreased caspase-8 activity in 6-OHDA-induced PC12 cells. Hence, isovitexin (2), luteolin-7-O-β-d-glucopyranoside (4), and (+)-catechin (8) protected PC12 cells from 6-OHDA-induced apoptotic neurotoxicity.     

Evaluation of antiprotozoal and antimycobacterial activities of the resin glycosides and the other metabolites of Scrophularia cryptophila

Resin glycosides are secondary metabolites exclusive to the convolvulaceous plants. In this study, crypthophilic acids A–C (1–3), the first resin glycosides occurring in another family (Scrophulariaceae), and the other constituents of Scrophularia cryptophila were examined for in vitro antiprotozoal and antimycobacterial potentials. Except for crypthophilic acid B (2), all tested compounds exhibited growth-inhibitory effect against Trypanosoma brucei rhodesiense, with l-tryptophan (6) and buddlejasaponin III (7) being the most potent ones (IC₅₀'s 4.1 and 9.7 μg/ml). In contrast, the activity towards Trypanosoma cruzi was poor, and only crypthophilic acid C (3), 6 and 7 were trypanocidal at concentrations above 40 μg/ml. With the exception of 2 and 6, all compounds were active against Leishmania donovani. Harpagide (4) and 3 emerged as the best leishmanicidal agents (IC₅₀'s 2.0 and 5.8 μg/ml). Only compounds 3, 6 and 7 showed antimalarial activity against Plasmodium falciparum with IC₅₀ values of 4.2, 16.6 and 22.4 μg/ml. Overall the best and broadest spectrum activity was presented by compounds 3 and 7, as they inhibited all four parasitic protozoa. None of the isolates had significant activity against Mycobacterium tuberculosis (MICs >100 μg/ml) or were toxic towards mammalian (L6) cells. This is the first report of antiprotozoal activity for natural resin glycosides, as well as for harpagide (4), acetylharpagide (5), tryptophan (6) and buddlejasaponin III (7).     

Inhibitory effects of ethyl acetate extract of Cordyceps sinensis mycelium on various cancer cells in culture and B16 melanoma in C57BL/6 mice

The cultivated mycelium of a Cordyceps sinensis (Cs) fungus was sequentially extracted by petroleum ether (PE), ethyl acetate (EtOAc), ethanol (EtOH) and hot water. All solvent extracts except hot water extract showed a significant and dose-dependent inhibitory effect on the proliferation of four cancer cell lines, MCF-7 breast cancer, B16 mouse melanoma, HL-60 human premyelocytic leukemia and HepG2 human hepatocellular carcinoma, with IC(50) values below 132 microg/ml. The EtOAc extract, in particular, had the most potent effect against all four cancer cell lines, with IC(50) between 12 microg/ml (on B16) and 45 microg/ml (on MCF-7). In contrast, it had much lower cytotoxicity against normal mouse bone marrow cells. The EtOAc extract contained carbohydrates, adenosine, ergosterol and trace amount of cordycepin, of which ergosterol and related compounds were identified as a major class of active constituents contributing to the in vitro cytotoxicity. In an animal test, the EtOAc extract showed significant inhibiting effect on B16-induced melanoma in C57BL/6 mice, causing about 60% decrease of tumor size over 27 days. Our results suggest that the EtOAc extract of Cs fungal mycelium has strong anti-tumor activity and is a potential source of natural anti-tumor products.     

Lactobacillusacidophilus S-layer protein-mediated inhibition of Salmonella-induced apoptosis in Caco-2 cells

Surface layer (S-layer) proteins are crystalline arrays of proteinaceous subunits present as the outermost component of the cell wall in several Lactobacillus species. The underlying mechanism for how S-layer proteins inhibit pathogen infections remains unclear. To gain insights into the mechanism of the antimicrobial activity of Lactobacillus S-layer proteins, we examined how Lactobacillus S-layer proteins impact Salmonella Typhimurium-induced apoptosis in vitro in Caco-2 human colon epithelial cells. When Caco-2 cells infected with Salmonella Typhimurium SL1344, we found that apoptosis was mediated by activation of caspase-3, but not caspase-1. When Salmonella Typhimurium SL1344 and S-layer proteins were coincubated simultaneously, Caco-2 cell apoptosis was markedly decreased and the cell damage was modified, as evaluated by flow cytometry and microscopy. Detailed analyses showed that the S-layer proteins inhibited the caspase-3 activity and activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway. Taken together, these findings suggest that Lactobacillus S-layer proteins protected against Salmonella-induced apoptosis through reduced caspase-3 activation. In addition, Salmonella-induced apoptotic cell damage was modified by S-layer proteins through the ERK1/2 signaling pathway. This mechanism may represent a novel approach for antagonizing Salmonella infection.