Volume 65, Issue 7-8

The water-accumulating leaves of crassulacean acid metabolism plants belonging to five different families were investigated for the presence of biominerals by infrared spectroscopic and microscopic analyses. Spectroscopic results revealed that the mineral present in succulent species of Agavaceae, Aizoaceae, and Asphodelaceae was calcium oxalate monohydrate (whewellite, CaC 2 O 4 · H 2 O). Crystals were predominantly found as raphides or solitary crystals of various morphologies. However, representative Crassulaceae members and a succulent species of Asteraceae did not show the presence of biominerals. Overall, these results suggest no correlation between calcium oxalate generation and crassulacean acid metabolism in succulent plants.

Several 2,6-bis-(un)substituted phenoxymethylpyridines were synthesized and evaluated in vitro against Fusarium graminearum, Helminthosporium sorokinianum, Alternaria brassicae, Alternaria alternata, and Fusarium oxysporum f. sp. vasinfectum. Among all derivatives, compound 3 a exhibited a broad-spectrum antifungal activity against the five phytopathogenic fungi.

Nine indole derivatives were evaluated in vitro against Fusarium graminearum, Alternaria alternata, Helminthosporium sorokinianum, Pyricularia oryzae, Fusarium oxysporum f. sp. vasinfectum, Fusarium oxysporum f. sp. cucumarinum, and Alternaria brassicae. Most of the compounds were found to possess antifungal activities. Especially compounds 2, 5, 8, and 9 exhibited broad-spectrum antifungal activities against the above-mentioned seven phytopathogenic fungi, and showed more potent activities than hymexazole, a commercial agricultural fungicide.

Application of acetylcholinesterase (AChE) inhibitors is the primary treatment for Alzheimer’s disease. Alkaloids, such as physostigmine, galanthamine, and huperzine A, play an important role as AChE inhibitors. The aim of this work was to evaluate Himatanthus lancifolius (Muell. Arg.) Woodson, a Brazilian species of Apocynaceae, and its main indole alkaloid uleine, in order to identify new AChE inhibitors. The plant fluid extract, fractions, and uleine were tested for AChE inhibitory activity using Ellman’s colorimetric method for thin-layer chromatography (TLC), 96-well microplates, and also Marston’s TLC colorimetric method. Both TLC assays showed similar results. At 5 mg/mL, the fluid extract inhibited the AChE enzyme by (50.71 ± 8.2)%. The ethyl acetate fraction exhibited the highest level of AChE inhibition, followed by the dichloromethane fraction. The isolated alkaloid uleine displayed an IC 50 value of 0.45 μM.

Hyaluronidase inhibitors are of potential therapeutic value for the treatment of a variety of diseases, such as cancer, arthrosis, or bacterial infections. Potent and selective hyaluronidase inhibitors are not known so far, and current approaches to the development of hyaluronidase inhibitors are limited. Elevated levels of hyaluronan (HA) are connected with most malignant tumours. Therefore, the search for drugs modulating the hyaluronidase activity became very important. In the present study, a new series of aminomethyl indole derivatives (AMIDs) were tested for inhibition of bovine testes hyaluronidase (BTH). In vitro assays were performed using stains-all at pH 7 and Morgan-Elson reaction at pH 3.5. Among the AMIDs, 3-[(4-methylpiperazin-1-yl)methyl]-5-phenyl-1H-indole (9) was found to be active with 23% inhibition at 50 μM and pH 7. All the other inhibitors showed less activity at pH 3.5 and pH 7. These activity results demonstrated that compounds with phenyl substitution at position 5 have higher activity. The results confirmed that more lipophilic compounds have better inhibition against the hyaluronidase enzyme.

The vasorelaxant response of N-p-nitrophenylmaleimide (4-NO 2 -NPM) was evaluated. The mesenteric rings (1 - 2 mm i.d.) were suspended by cotton thread for isometric tension recordings in a Tyrode’s solution at 37 °C and gassed with a mixture of 95% O 2 and 5% CO 2 , under a resting tension of 0.75 g. 4-NO 2 -NPM induced relaxation in mesenteric rings pre-contracted with phenylephrine (Phe; 10 μM, pD2 = 6.7 ± 0.3) or KCl (80 mM, pD2 = 3.9 ± 0.2). This effect was significantly attenuated after removal of the vascular endothelium, N G -nitro L-arginine methyl ester (L-NAME; 100 μM), atropine (1 μM), indomethacin (10 μM), L-NAME + indomethacin or 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ; 10 μM). LArginine (1 mM) reversed the inhibitory effect of L-NAME. In endothelium-intact preparations pre-incubated with 20 mM KCl, tetraethylammonium bromide (TEA; 1 mM) or glibenclamide (Glib; 10 μM), the vasorelaxant effect was significantly attenuated when compared to controls (endothelium intact). In denuded rings, separate incubation with 20 mM KCl, TEA or Glib did not change the relaxation when compared with that obtained in denuded rings. 4-NO 2 -NPM inhibited in a concentration-dependent and non-competitive manner the concentration-response curves induced by CaCl 2 . In calcium-free medium, the transient contractions induced by Phe (10 μM) or caffeine (20 mM) were inhibited. The relaxant effect induced by 4-NO 2 -NPM appeared to be due to endothelial muscarinic receptors activation, NO and prostacyclin release and KATP and BKCa (Ca 2+ -activated K + channels) endotheliumdependent activation. Inhibition of the Ca 2+ influx and inhibition of the Ca 2+ release from intracellular IP 3- and caffeine-sensitive stores are also involved in the vasorelaxation

The galanthamine, lycorine, and lycoramine content of Lycoris chinensis was researched during development from young to old plants, i.e. in seeds, ten-day-old seedlings, threemonth- old seedlings, one-year-old seedlings, and perennial seedlings. Notably the alkaloid level reduced to its lowest content 10 days after seed germinating. Then the accumulation of galanthamine tended to increase with age, reaching a higher value in perennial seedlings. The production pattern of lycorine and lycoramine was found similar to that of galanthamine. Different plant organs were also evaluated for their galanthamine, lycorine, and lycoramine contents. Mature seeds had the highest content of galanthamine (671.33 μg/g DW). Kernels, seed capsules, and root-hairs were the main repository sites for galanthamine, lycorine, and lycoramine. The leaves were the least productive organs.

The toxic effects of H 2 S on plants are well documented. However, the molecular mechanisms reponsible for inhibition of plants by H 2 S are still not completely understood. We determined the effects of NaHS in the range of 0.5 - 10 mM on the growth of rice suspension culture cells, as well as on the expression of the alternative oxidase (AOX) gene. AOX is the terminal oxidase of the alternative pathway (AP) and exists in plant mitochondria. The results showed that H 2 S treatment enhanced the AP activity. During the process of H 2 S treatment for 4 h, the AP activity increased dramatically and achieved the peak value at a concentration of 2 mM NaHS. Then it declined at higher concentrations of NaHS (5 - 10 mM) and maintained a steady level. The AOX1 gene transcript level also showed a similar change as the AP activity. Interestingly, different NaHS concentrations seemed to have different effects on the expression of AOX1a, AOX1b, and AOX1c. The induction of AOX expression by low concentrations of NaHS was inferred through a reactive oxygen species (ROS)-independent pathway. At the same time, rice cells grown in culture were very sensitive to H 2 S, different H 2 S concentrations induced an increase in the cell viability. These results indicate that the H 2 S-induced AOX induction might play a role in inhibiting the ROS production and have an influence on cell viability.

Phenylethanoids are a group of natural water-soluble compounds with high biological value, which could potentially be commercially produced by hairy root cultures. Thus, we have examined the capacity of transformed root cultures of Devil’s claw (Harpagophytum procumbens) to accumulate four phenylethanoid glycosides - β-OH-verbascoside, verbascoside, leucosceptoside A, and martynoside - in shake-flasks and a 3-L stirred tank reactor. Verbascoside was found to be the major phenylethanoid, and its maximal contents were the same (1.12 mg/g dry weight) in both kinds of culture. However, peak leucosceptoside A contents were 1.6-times higher in bioreactor cultures than in shake-flask cultures. Flow cytometry analysis revealed that G 0 + G 1 -phase cells predominated throughout the growth of the cultures, which was in accordance with the very high proportion of quiescent cells in the transformed roots. The results provide the first demonstration of the potential utility of Devil’s claw hairy roots as biofactories for producing high-value phenylethanoid glycosides.

In most studies dealing with the laboratory cultivation of paramecia (Paramecium bursaria), Klebsiella pneumoniae bacteria are used to inoculate the medium. However, Klebsiella pneumoniae is a typical pathogen, and its use is always associated with a risk of infection. The aim of the present research was to examine non-pathogenic bacteria strains as components of the medium for Paramecium bursaria. The paramecia were incubated on lettuce infusions bacterized with different bacteria strains: Bacillus subtilis DSM 10, Bacillus megaterium DSM 32, Escherichia coli DSM 498, Micrococcus luteus DSM 348. A strain derived from the natural habitat of Paramecium bursaria was used as the control one. Experiments were conducted under constant light and in the dark. Paramecia cells were counted under a stereomicroscope on consecutive days of incubation. The obtained results show that the most intensive growth of Paramecium bursaria occurs in the presence of Escherichia coli DSM 498. The use of this strain as a component of the medium allows one to obtain a high number of ciliates regardless of the light conditions. It can be concluded that the Paramecium bursaria cultivation procedure can be modified by using the non-pathogenic bacteria strain Escherichia coli DSM 498 instead of Klebsiella pneumoniae.

Lipases are enzymes used in various industrial sectors such as food, pharmaceutical and chemical synthesis industries. The selection of microorganisms isolated from soil or wastewater is an alternative to the discovery of new species with high enzymes productivity and with different catalytic activities. In this study, the selection of lipolytic fungi was carried out by submerged fermentation. A total of 27 fungi were used, of which 20 were isolated from dairy effluent and 7 from soil contaminated with diesel oil. The largest producers were the fungi Penicillium E-3 with maximum lipolytic activity of 2.81 U, Trichoderma E-19 and Aspergillus O-8 with maximum activities of 2.34 and 2.03 U where U is the amount of enzyme that releases 1 μmol of fatty acid per min per mL of enzyme extract. The fungi had maximum lipolytic activities on the 4th day of fermentation.

This study presents an HPLC method for simultaneous analysis of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous. The HPLC method is accomplished by employing a C 18 column and the mobile phase methanol/water/acetonitrile/ dichloromethane (70:4:13:13, v/v/v/v). Astaxanthin is quantified by detection at 480 nm. The carotenoid precursors are identified by LC-APCI-MS and UV-vis absorption spectra. Peaks showed in the HPLC chromatogram are identified as carotenoids in the monocyclic biosynthetic pathway or their derivatives. In the monocyclic carotenoid pathway, 3,3’-dihydroxy- β,ψ-carotene-4,4’-dione (DCD) is produced through γ-carotene and torulene.

Genetic relationships were studied among eight species of three taxa in the genus Chaenomeles by nuclear ribosomal internal transcribed spacer (ITS) analysis. A genetic distance matrix based on ITS sequences was estimated according to the formula of Kimura-2 parameter and a neighbour-joining phenogram, which were obtained with Clustalx4.1 software. The results showed that the germplasms of Mugua originate from Ch. speciosa (Sweet) Nakai, not including Ch. sinensis (Thouin) Kochne and Ch. cathayensis (Hemsl.) Schneid. The results also showed that ‘Yao Mugua’ and ‘Ornamental Mugua’ are the most distantly related species in germplasms.

A novel 1 L-myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4) gene, designate rcMIPS, was cloned from Ricinus communis. It contained an open reading frame (ORF) of 1669 bp coding for a peptide of 510 amino acids with a molecular mass of 56 kDa. Sequence anaylsis showed high homology compared to other plant MIPS genes, because it contained typical domains owned by MIPS enzymes. The transcript levels of the rcMIPS gene in leaves, stems, and roots were examined after drought stress for 24, 48, and 72 h. The transcript levels in the leaves, stems, and roots increased significantly compared to the control. Results of the enzyme assay showed a significant correlation between the changes of enzyme activity and the transcript levels of the rcMIPS gene in different organs. Decreased relative water contents (RWC) and increased malondialdehyde (MDA) contents in the leaves represented a stress response against drought stress. Our findings suggest that MIPS plays an important role in the defensive mechanisms of R. communis against drought stress.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF5 (Bm5) is a gene present in many lepidopteran nucleopolyhedroviruses (NPVs), but its function is unknown. In this study, Bm5 was characterized. The transcript of Bm5 was detected 12 - 72 h post infection (p.i.). Polyclonal antiserum raised to a His-BM5 fusion protein recognized BM5 in infected cell lysates from 24 to 72 h p.i., suggesting that Bm5 is a late gene. Immunofluorescence analysis by confocal microscopy showed that the BM5 protein is localized primarily in the cytoplasm. Localization of BM5 in budded virion (BV) and occlusion-derived virion (ODV) by Western analyses demonstrated that BM5 is not a structural protein associated with BV or ODV.

The ornithine decarboxylase antizyme inhibitor (AZI) was discovered as a protein that binds to the regulatory protein antizyme and inhibits the ability of antizyme to interact with the enzyme ornithine decarboxylase (ODC). Several studies showed that the AZI protein is important for cell growth in vitro. However, the function of this gene in vivo remained unclear. In our study, we analyzed the transcriptional profiles of livers on the 19th day of pregnancy of Azin1 knock-out mice and wild-type mice using the Agilent oligonucleotide array. Compared to the wild-type mice, in the liver of Azin1 knock-out mice 1812 upregulated genes (fold change ≥ 2) and 1466 downregulated genes (fold change ≤ 0.5) were showed in the microarray data. Altered genes were then assigned to functional categories and mapped to signaling pathways. These genes have functions such as regulation of the metabolism, transcription and translation, polyamine biosynthesis, embryonic morphogenesis, regulation of cell cycle and proliferation signal transduction cascades, immune response and apoptosis. Real-time PCR was used to confirm the differential expression of some selected genes. Overall, our study provides novel understanding of the biological functions of AZI in vivo.

Cultural factors affecting the production of L-asparaginase by Streptomyces tendae isolated from laterite soil samples of Guntur region were investigated on glycerolasparagine- salts (modified ISP-5) broth. Optimal yields of L-asparaginase were recorded in the culture medium with the initial pH 7.0 incubated at 30 °C for 72 h. The strain utilized sucrose (2%) and yeast (2%) extract as carbon and nitrogen sources for L-asparaginase production. The productivity of L-asparaginase was slightly enhanced when the strain was treated with cell-disrupting agents like EDTA. The crude enzyme was purifi ed to homogeneity by ammonium sulfate precipitation, Sephadex G-100 and CM-Sephadex G-50 gel filtration. By employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was recorded as 97.4 kDa. This is the first report on production and purification of L-asparaginase from S. tendae.