In vitro antiplasmodial activity of extract and constituents from Esenbeckia febrifuga, a plant traditionally used to treat malaria in the Brazilian Amazon

Esenbeckia febrifuga (Rutaceae) is a plant traditionally used to treat malaria in the Brazilian Amazon region. Ethanol extract of stems displayed a good antiplasmodial activity against Plasmodium falciparum strains W-2 (IC₅₀ 15.5±0.71 μg/ml) and 3 D7 (IC₅₀ 21.0±1.4 μg/ml). Two coumarins (bergaptene 1 and isopimpinellin 2), five alkaloids (flindersiamine 3, kokusaginine 4, skimmiamine 5, γ-fagarine 6 and 1-hydroxy-3-methoxy-N-methylacridone, 7), besides a limonoid (rutaevine 8), have been isolated for the first time from this species. Antiplasmodial activity of compounds 3, 5–8 has been evaluated in vitro against P. falciparum strains (W-2 and 3D7) and the furoquinolines 5 and 6 were the most potent displaying IC₅₀ values <50 μg/ml; flindersiamine (3) showed a weak activity while alkaloid 7 and rutaevine (8) were inactive (IC₅₀>100 μg/ml).     

A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria

A 125-member library of synthetic linear undecapeptides was prepared based on a previously described peptide H-K¹KLFKKILKF¹⁰L-NH₂ (BP76) that inhibited in vitro growth of the plant pathogenic bacteria Erwinia amylovora, Xanthomonas axonopodis pv. vesicatoria, and Pseudomonas syringae pv. syringae at low micromolar concentrations. Peptides were designed using a combinatorial chemistry approach by incorporating amino acids possessing various degrees of hydrophobicity and hydrophilicity at positions 1 and 10 and by varying the N-terminus. Library screening for in vitro growth inhibition identified 27, 40 and 113 sequences with MIC values below 7.5 μM against E. amylovora, P. syringae and X. axonopodis, respectively. Cytotoxicity, bactericidal activity and stability towards protease degradation of the most active peptides were also determined. Seven peptides with a good balance between antibacterial and hemolytic activities were identified. Several analogues displayed a bactericidal effect and low susceptibility to protease degradation. The most promising peptides were tested in vivo by evaluating their preventive effect of inhibition of E. amylovora infection in detached apple and pear flowers. The peptide H-KKLFKKILKYL-NH₂ (BP100) showed efficacies in flowers of 63–76% at 100 μM, being more potent than BP76 and only less effective than streptomycin, currently used for fire blight control.     

Relationships between kernel vitreousness and dry matter degradability for diverse corn germplasm: II. Ruminal and post-ruminal degradabilities

Correlations between kernel vitreousness and ruminal in situ (RDMD) and total tract dry matter (TDMD; sum of ruminal in situ and post-ruminal in vitro measurements) degradabilities were determined for 33 diverse corn germplasm sources. These included a wide range of endosperm characteristics from opaque 2 (o2) types to densely packed flint types, and a number of intermediates. Harvests were done at two growth stages; 1/2 milk-line (ML) and black-layer (BL). Kernels from middle portion of ears were oven dried at 40 °C for 72 h and ground through a Wiley mill (6 mm screen) for measurement of in situ RDMD after 0 and 14 h of incubation using two steers (1.5 g/bag × 8 replicates per time point per steer in 5 cm × 5 cm bags of 50 μm pore size). Residue from the 14 h bags proceeded to an 8 h in vitro enzymatic post-ruminal digestion after which the residue was oven dried at 62 °C for 48 h and dry matter content determined. Inbred by harvest-stage interactions were observed for 0-h disappearance and TDMD. Vitreousness had strong negative correlations with degradability measurements, particularly for more mature (BL) samples (−0.728, −0.770 and −0.603) versus ML (−0.569, −0.541 and −0.338) for 0 h disappearance, RDMD and TDMD, respectively. Vitreousness was highly correlated with corn degradability, especially at a black-layer stage of harvest, in this diverse corn germplasm.     

Protective effect of Ecklonia cava enzymatic extracts on hydrogen peroxide-induced cell damage

In this study, Ecklonia cava was enzymatically hydrolyzed to prepare water-soluble extracts, using five carbohydrases (Viscozyme, Celluclast, AMG, Termamyl, and Ultaraflo) and five proteases (Protamex, Kojizyme, Neutase, Flavourzyme, and Alcalase), and the potential antioxidant activity of each was assessed. The Celluclast and Viscozyme extracts of E. cava evidenced good hydrogen peroxide (H₂O₂) scavenging activities (73.25% and 72.92%, respectively) as compared to those of other enzymatic extracts. Therefore, the Celluclast enzymatic extract was selected for use in further experiments, and separated into four different molecular weight fractions (<1, 1–10, 10–30 and >30 kDa). Among these fractions, the >30 kDa fraction manifested the most profound H₂O₂ scavenging activity, with a measured IC₅₀ of 13 μg/ml. The >30 kDa fraction also strongly enhanced cell viability against H₂O₂-induced oxidative damage, and evidenced relatively good lipid peroxidation inhibitory activity in a Chinese hamster lung fibroblast (V79-4) cell line. This fraction also effected a reduction in the proportion of cells undergoing H₂O₂-induced apoptosis, as was demonstrated by a decreased quantity of sub-G₁ hypodiploid cells and decreased apoptotic body formation on the flow cytometry assay. These results clearly indicate that the >30 kDa fraction of E. cava possesses good antioxidant activity against H₂O₂ mediated cell damage in vitro.     

In vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces

The in vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces were studied by means of light and electron microscopy. Both drugs had a protoscolicidal activity that increased proportionally with increasing concentrations of the drugs. Ivermectin showed the more rapid effects and caused contraction and paralysis of protoscoleces. A paralyzing effect was also observed with levamisole, followed by irreversible tissue vacuolation leading to death.     

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis

Ecdysterone induced sexual reproduction in Opalina sudafricana parasitic in Bufo regularis. International Journal for Parasitology 3: 863–868. Ecdysterone injections (0·5 mg/ml) induced sexual reproduction in Opalina sudafricana parasitic in male or female Bufo regularis after 12 days. Hypophysectomy or gonadectomy did not prevent the hormone from producing its effect on the parasites. Ecdysterone did not induce sexual reproduction in the parasites in vitro. Urine of toads injected with ecdysterone induced the opalinids to encyst in vitro.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Monoquaternary ammonium derivatives inhibit growth of protozoan parasites

The phospholipid metabolism of Plasmodium falciparum-infected erythrocytes has been shown to be an effective pharmacological target for novel chemotherapy. Thirty-seven monoquaternary ammonium derivatives analogous to choline were screened for their potential antiprotozoal activity against P. falciparum and Leishmania braziliensis. Twenty-three compounds inhibited chloroquine resistant and sensitive P. falciparum strains with inhibitory concentrations ranging from 0.001 μM to 47 μM. Among the inhibitors were six compounds with nanomolar activity containing at least one ethyl group in the polar head and a hydrophobic alkyl chain with 10 to 14 methylene groups. Four compounds also exhibited in vitro antileishmanial properties in the micromolar range.     

Effects of ultrashort gamete co-incubation time on porcine in vitro fertilization

A reduction in co-incubation time has been suggested as an alternative method to reduce polyspermic fertilization. The aim of this study was to evaluate the effect of short periods of gamete co-incubation during pig in vitro fertilization. A total of 2833 in vitro matured oocytes were inseminated with thawed spermatozoa and coincubated for 0.25, 1, 2, 3, 7, 10 min and 6 h. The oocytes from the 0.25–10 min groups were washed three times in modified Tris-buffered medium (mTBM) medium to remove spermatozoa not bound to the zona and transferred to the same medium (containing no spermatozoa) until 6 h of co-incubation time were completed. After 6 h, presumptive zygotes from each group were cultured in NCSU-23 medium for 12–15 h to assess fertilization parameters. After each period of co-incubation, 45–50 oocytes from each group were stained with Hoechst-33342 and the number of spermatozoa bound to the zona was counted. Although the number of zona bound spermatozoa increased (p < 0.05) with the co-incubation time, no increase was observed in penetration rates among groups from 2 min to 6 h of co-incubation time (ranging from 53.5 ± 2.8 to 61.3 ± 2.6%). Similarly, the efficiency of fertilization reached a maximum for the 2 min of co-incubation group with values ranging between 32.3 ± 2.4 and 41.9 ± 2.5%. The reduction of co-incubation time did not affect the monospermy rate (range: 71.3 ± 3.4–80.2 ± 3.8%) and the mean number of spermatozoa/oocyte (range: 1.2 ± 0.4–1.4 ± 0.5). These results show that, under our in vitro conditions, high penetration rate can be obtained with co-incubation times as short as 2 min, although monospermy could not be improved using this strategy.     

Freezing resistance improvement of Lactobacillus reuteri by using cell immobilization

Lactobacillus reuteri shows certain beneficial effects to human health and is recognized as a probiotic. However, its application in frozen foods is still not popular because of its low survival during freezing and frozen storage. Cell immobilization technique could effectively exert protection effects to microbial cells in order to enhance their endurance to unfavorable environmental conditions as well as to improve their viability and cell concentration. Ca-alginate and κ-carrageenan were used to immobilize L. reuteri in this research, and the immobilized cells were exposed to different freezing temperatures, i.e. − 20 °C, − 40 °C, − 60 °C, − 80 °C, and stored at − 40 °C and − 80 °C for 12 weeks. The objectives were to study the protection effects of cell immobilization against the adverse conditions of freezing and frozen storage, and the effects of freezing temperatures to the immobilized cells. Cell immobilization was used to raise the survival of L. reuteri during freezing and frozen storage in order to develop frozen foods with the probiotic effects of L. reuteri. Results indicated that immobilized L. reuteri possessed better survival in both freezing and frozen storage. The survival of immobilized L. reuteri was higher than that of free cells, and the effects of lower freezing temperature were better than higher freezing temperature. The immobilization effects of Ca-alginate were found to be superior to κ-carrageenan. Cell immobilized L. reuteri exhibits potential to be used in frozen foods.     

Molecular characterization of a novel gene encoding an 8-kDa-subunit of antigen B from Echinococcus granulosus genotypes 1 and 6

Antigen B in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa, and is an aggregate of several different but homologous small proteins with approximately 8 kDa which are encoded by a multigene family. Four genes encoding 8-kDa-subunit monomers of the antigen B have been identified from E. granulosus. Recently, we have isolated another novel gene from Echinococcus multilocularis encoding a fifth 8-kDa-subunit of AgB (named EmAgB8/5), predominantly transcribed in the adult worm, but not in vesicles of metacestodes. In this study, we cloned and characterized two EmAgB8/5 homologue genes from E. granulosus genotypes 1 and 6 by PCR, and named as EgG1AgB8/5 and EgG6AgB8/5, respectively. The phylogenetic relationship of these genes with other genes encoding the antigen B 8-kDa-subunit monomers was also discussed.     

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.

Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.

The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.

Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.

Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).

These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.     

A simple in-vitro 'wet-plate' method for mass production of Phytophthora nicotianae zoospores and factors influencing zoospore production

A simple in-vitro ‘wet-plate’ method for mass-producing Phytophthora nicotianae zoospores at ≥ 1.0 × 10⁶ zoospores/ml is described. Temperature critically affected zoospore production; 22 °C was optimum, while 36 °C was completely inhibitory. Zoospores being the most important propagule of P. nicotianae, temperature of recycled irrigation water may be manipulated to reduce diseases in irrigated nursery crops.     

Phosphoramidon potentiates the endothelin-1-induced bronchopulmonary response in guinea-pigs

We investigated the effect of the endopeptidase-inhibitor, phosphoramidon, on the bronchopulmonary response induced by endothelin-1 in vivo or in isolated perfused lungs. In vivo aerosol administration of 1 or 3 μg/ml endothelin-1 for 2 min provoked no significant bronchopulmonary response. When awake animals were pretreated by an aerosol of phosphoramidon (0.1 mM, for 15 min), the bronchopulmonary response induced by 1 and 3 μg/ml endothelin-1 was markedly enhanced. In isolated guinea-pig lungs, aerosol administration of endothelin-1 (3 μg/ml, for 2 min) evoked a low increase in pulmonary inflation pressure. Treatment of awake animals with an aerosol of phosphoramidon before lung recollection led to a significant potentiation of the endothelin-1-induced increase in pulmonary inflation pressure. These results demonstrate that phosphoramidon potentiates the in vivo and in vitro bronchopulmonary response evoked by low doses of endothelin-1 and suggest that endopeptidase-like enzymes present in the airway tissue modulate the effect of the peptide.     

Determination of the thermodynamics of carbonic anhydrase acid-unfolding by titration calorimetry

The enthalpy of unfolding (Δ_uH) of carbonic anhydrase II was determined by titrating the protein with acid and measuring the heat using isothermal titration calorimetry (ITC) in the temperature range of 5 to 59 °C. By combining the ITC results with our previous findings by differential scanning calorimetry (DSC) in the temperature range of 39 to 72 °C, the Δ_uH dependence over a wide temperature range was obtained. The temperature dependence of the enthalpy displays significant curvature indicating that the heat capacity of unfolding (Δ_uC_p) is dependent on temperature. The T-derivative of Δ_uC_p was equal to 100 ± 30 J/(mol × K²), with the result that the Δ_uC_p is equal to 15.8 kJ/(mol × K) at 5 °C, 19.0 kJ/(mol × K) at 37 °C and 21.8 kJ/(mol × K) at 64 °C. The enthalpy of unfolding is zero at 17 °C. At lower temperatures, the Δ_uH becomes exothermic.

This method of determining protein unfolding thermodynamics using acid-ITC, significantly widens the accessible T-range, provides direct estimate of the thermodynamic parameters at physiological temperature, and gives further insight into the third T-derivative of the Gibbs free energy of unfolding.     

The anticarcinogenic effect of propolis in human lymphocytes culture

The in vitro anticarcinogenic potential of propolis in human lymphocytes was investigated. Blood samples were obtained from ten healthy males, non-smoking volunteers, which were incubated and exposed to increasing concentrations of propolis (0.01, 0.05, 0.1, 0.2, 0.5, 0.7 and 1.0 ml). The mean micronucleus rates were 1.47±0.38–4.02±0.64. Mitotic index rates were between 19.45±2.22 and 0.28±0.33. The differences between the control and exposed cells were statistically significant (p0.05). We conclude that exposure to different concentrations of propolis cannot produce a carcinogenic effect in peripheral human lymphocytes in vitro. However, increasing micronucleus (MN) rates showed that propolis could have a carcinogenic effect in high concentrations.

Also chemical analysis of propolis sample was evaluated by GC/MS. Propolis sample mainly contains flavonoids, fatty and aromatic acids and their esters.     

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.     

Association of the 90-kDa heat shock protein does not affect the ligand-binding ability of androgen receptor

An N-terminal truncated androgen receptor with putative DNA- and ligand- binding domains (AR438) and that with a ligand-binding domain (AR612) were expressed under control of the T7 promoter in E. coli or translated in vitro with rabbit reticulocyte lysate, and their ligand-binding properties and the interaction with HSP90 were investigated. Bacterially expressed AR438 and AR612 bound a synthetic androgen, [³H]R1881, with apparent dissociation constant of 2.6 ± 0.2 and 3.1 ± 0.7 nM, respectively, values which are comparable to those of androgen receptor in target tissues. The recombinant androgen receptors sedimented at the 4–5 S region irrespective of the presence of 10 mM tungstate, indicating that the receptor exists free from HtpG, which is the bacterial homolog of eukaryotic HSP90. The apparent dissociation constant of truncated androgen receptors translated in vitro was 0.1 nM for AR438 and 0.2 nM for AR612. Sedimentation coefficients of in vitro translated molecules were converted from 7–8 S in the presence of tungstate to 3 S in the absence of tungstate. Both AR438 and AR612 translated in vitro were retained by anti-rat HSP90 antibody-protein A Sepharose. Exposure to 0.3 M NaCl in the presence of ligand caused dissociation of AR438 and AR612 from HSP90, and concomitantly, the DNA-cellulose binding ability of AR438 was enhanced. Thus, we conclude that the androgen receptor associates with HSP90 through the ligand-binding domain and that this association prevents the interaction of the androgen receptor with DNA. However, HSP90 seems to have little effect on the ligand-binding characteristics of the androgen receptor.