Simple method for separating the chlamydoconidia of Candida albicans from its mycelium

Different physical and chemical methods were used to detach the chlamydoconidia of Candida albicans from its mycelium. The action of concentrated H₂SO₄ acid for a 4-min period on cultures lysed both the mycelium and the outer but not the inner wall layer of the chlamydoconidia.The sulfuric acid procedure is recommended as the best method to obtain mycelium free chlamydoconidia because of its simplicity, rapidity and low cost.     

Bikaverin,an antibiotic fromGibberella fujikuroi,effective againstLeishmania brasiliensis

The red quinone C₂₀H₁₄O₈ (mp 322–324), named bikaverin was isolated from mycelium ofGibberella fujikuroi. Bikaverin is specifically effective againstLeishmania brasiliensis while is not effective against other protozoa, bacteria and fungi. For production of bikaverin in a submerged culture the most convenient media are those with a natural source of nitrogen (corn-steep liquor, soyabean meal) in which the content of bikaverin ranged from 1.0 to 1.7% of dry weight of mycelium.     

Effects of the glutamine synthetase inhibitor methionine sulfoximine on CO2 fixation inLemna gibba

Summary Net CO₂ fixation inLemna gibba L. was inhibited by 0.5 mM L-methionine D,L-sulfoximine (MSX) both under photorespiratory conditions (21% O₂) and in 2% O₂. The inhibition was noticeably delayed by addition of 5 mM glutamine. Glutamine also delayed MSX-induced inactivation of glutamine synthetase. An increase in intracellular NH ₄ ⁺ concentration was noted in the presence of MSX only, and in the presence of 10 mM NH ₄ ⁺ only. However, presence of 10 mM NH ₄ ⁺ did not cause any inhibition of CO₂ fixation.     

Tuber-forming Substances in Jerusalem Artichoke (Helianthus tuberosus L.)

Four tuber-forming substances in Jerusalem artichoke were isolated from the leaves. The structures were established by spectroscopic methods as jasmonic acid (2), methyl β-D-glucopyranosyl tuberonate (3), and two new polyacetylene compounds, methyl β-D-glucopyranosyl helianthenate A (4, C₁₉H₂₄O₈) and B (5, C₁₇H₂₂O₈).     

A Non-radioisotopic Anion-exchange Chromatographic Method to Measure the CO2/O2 Specificity Factor for Ribulose Bisphosphate Carboxylase/Oxygenase

A non-radioisotopic anion-exchange ion chromatographic method for measuring the carboxylation/ oxygenation specificity (τ) of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is presented. The assay measures the amounts of fixation products at varying [CO₂]/[O₂] ratios to measure the relative rates of CO₂ and O₂ fixation reactions. The amount of 3-phosphoglycerate (3-PGA) and phosphoglycolate (PG) in the reaction mixture were measured with a conductivity detector and the specific factor was calculated using the following equations: ν_c = ([3-PGA] – [PG])/2 and ν_o = [PG]. By this method, specificity factors for RubisCOs were measured without using radioactive reagents.     

SPATIAL SEGREGATION OF CO2 FIXATION IN TRICHODESMIUM SPP.: LINKAGE TO N2 FIXATION POTENTIAL1

The aggregate-forming, nonheterocystous, filamentous blue-green alga (cyanobacteria) Trichodesmium spp. is a widespread and important planktonic N₂ fixer and primary producer in tropical and subtropical oceans. It is unique among nonheterocystous genera because it conducts N₂ and CO₂ fixation (O₂ evolution) simultaneously; a notable achievement, because O₂ is a potent inhibitor of N₂ fixation. Spatial and temporal CO₂ fixation patterns were examined in trichomes and aggregates from natural and cultured populations, utilizing microautoradiographic detection of ¹⁴CO₂ incorporation. Parallel N₂ fixation (acetylene reduction) measurements were also made. Diel N₂ and CO₂ fixation patterns were similar, with co-optimization of both processes near midday. Microautoradiographs revealed several trichome-level ¹⁴CO₂ incorporation patterns: 1)uniform, heavy labeling, 2)uniform, light labeling, 3) heavier labeling in distal as opposed, to proximal regions, and 4) virtually no labeling throughout. Similar patterns were observed in natural and cultured populations. Given previous immunochemical findings that N₂ fixation potential is widespread in Trichodesmium spp. trichomes and aggregates, current results suggest a high degree of individuality, and possibly a “division of labor” in terms of CO₂ fixation, among trichomes comprising active N₂-fixing aggregates. Segregation of photosynthesis within and among trichomes facilitates simultaneous N₂ and CO₂ fixation in Trichodesmium spp. trichomes and aggregates.     

A reliable method with good cell preservation for the demonstration of peroxidase activity in human platelets and megakaryocytes

Summary We have tried to improve existing methods for demonstration of platelet peroxidase (PPO) in human platelets and megakaryocytes by introducing a fixation in 0.1% glutaraldehyde prior to incubation in the DAB medium. This prefixation with low concentration of glutaraldehyde preserves excellent morphological detail and does not inhibit PPO activity. All 23 platelet-rich plasma samples show PPO reaction product in the dense tubular system after incubation in DAB medium with 0.003% H₂O₂. When 0.01% H₂O₂ is used in excessive DAB medium, PPO activity can also be demonstrated in platelets and megakaryocytes of bone-marrow cell suspensions. This method can be used for the identification of megakaryoblasts in acute non-lymphocytic leukemia, myelodysplastic syndromes and in blastic crisis of chronic myeloid leukemia. PPO cytochemistry can be combined with postfixation in a OsO₄-ruthenium red mixture. This method reveals -granules, dense bodies, microtubul,, glycogen, mitochondria, dense tubular system and invaginated membrane system in the same platelet and is useful for investigation of platelet ultrastructure.     

Immunogold labeling of cryosections from high-pressure frozen cells

Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. A technique for ultracyotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–565), is an important and widely used method for immunoelectron microscopy. These sections are cut from material that is chemically fixed at room temperature (room temparature fixation, RTF). Lately in many morphological studies fast freezing followed by cryosubstitution fixation (CSF) is used instead of RTF. We have explored some new methods for applying immunogold labeling on cryosections from high‐pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). As immunolabeling has to be carried out on thawed and stable sections, we explored two ways to achieve this: (1) The section fixation method, as briefly reported before (Liou W et al. Histochem Cell Biol 1996;106:41–58 and Möbius W et al. J Histochem Cytochem 2002;50:43–55.) in which cryosections from freshly frozen cells were stabilized in mixtures of sucrose and methyl cellulose and varying concentrations of glutaraldehyde, formaldehyde and uranyl acetate (UA). Only occasionally does this method reveal section areas with excellent cell preservation and negatively stained membranes like Tokuyasu sections of RTF material. (Liou et al.) (2) The rehydration method, a novel approach, in which CSF with glutaraldehyde and/or osmium tetroxide (OsO₄) was followed by rehydration and cryosectioning as in the Tokuyasu method. Especially, the addition of UA and low concentrations of water to the CSF medium favored superb membrane contrast. Immunogold labeling was as efficient as with the Tokuyasu method.     

Production of gluconic acid by immobilized in pumice stones mycelium of Aspergillus niger using unconventional oxygenation of culture

Gluconic acid was produced in repeated batch processes with Aspergillus niger AM-11, immobilized in pumice stone particles using an unconventional oxygenation of culture media based on the addition of H₂O₂, decomposed by catalase to O₂ and water. The highest gluconic acid productivity of 8.2 g l^–1 h^–1 was reached with 30 g immobilized mycelium per 150 ml, 10% (w/v) glucose, at 24 °C and pH 6.5, with O₂ at 100% saturation. The immobilized mycelium was successfully reused up to 8 times in 1-h batches with only a slight loss (11%) of gluconic acid productivity.     

Effect of tissue processing on the immunostaining of inflammatory cell surface epitopes identified by monoclonal antibodies

Summary Optimal tissue processing conditions were defined for the immunohistochemical detection of inflammatory cell surface epitopes identified by OKT3, OKT4, OKT6, OKT8, OKIa1, OKM1, Leu7 and pan B cell antibodies. Snap-freezing in isopentan was superior to liquid nitrogen in preservation of morphology. Embedding of the tissues in Tissue-Tek II O.C.T. Compound diminished the intensity of immunostaining with all antibodies studied; however, the embedded tissues tolerated longer storage without drying. Optimal fixation with satisfactory preservation of morphology and immunogenicity was achieved with fixation of the frozen sections in acetone at 4° C for 5 min. Blocking of the endogenous peroxidase with methanol-H₂O₂ treatment destroyed all epitopes studied except those identified with OKIa1, OKT6 and Leu7.     

COLONIZATION OF AMERICA BY DROSOPHILA SUBOBSCURA: LETHAL GENES AND THE PROBLEM OF THE O5 INVERSION

In this work, the process of colonization of North and South America by the species Drosophila subobscura has been studied by analyzing the variability of lethal genes. The genetic structures of a Palearctic natural central population (Bordils, Spain) and a colonizer population from America (Gilroy, California) have been compared. The frequencies of lethal chromosomes and their allelism are 29.007% and 0.0069 in the first population and 14.414% and 0.0526 in the American population. A founder effect is detected after the computation of some population parameters (N_e, h, he and the lethal load). Furthermore, the allelism of lethal chromosomes has revealed a strong association between a lethal gene and the O₅ inversion both in Gilroy and in the population of Puerto Montt (Chile). The interpopulation allelism shows that the O₅ arrangement from the USA and Chile is the same, confirming that the colonizing processes of North and South America are correlated. The O₅ arrangement can also be useful as a genetic marker to trace the origin of the colonization. The frequency of the O₅ arrangement in the original population of the colonization could be used to estimate the number of colonizers. This population is still unknown, but taking the extreme values of the frequency of the O₅ inversion in natural Palearctic populations (1–15%), the number of colonizers could vary between 9 and 149 individuals.     

Thermally stimulated luminescence glow curve structure of β‐irradiated CaB4O7:Dy

Thermally stimulated luminescence glow curves of CaB₄O₇:Dy samples after β‐irradiation showed glow peaks at ~335, 530 and 675 K, with a heating rate of 2 K/s. The main peak at 530 K was analyzed using the T_max–T_stop method and was found to be composed of at least five overlapping glow peaks. A curve‐fitting program was used to perform computerized glow curve deconvolution (CGCD) analysis of the complex peak of the dosimetric material of interest. The kinetic parameters, namely activation energy (E) and frequency factor (s), associated with the main glow peak of CaB₄O₇:Dy at 520 K were evaluated using peak shape (PS) and isothermal luminescence decay (ILD) methods. In addition, the kinetics was determined to be first order (b =1) by applying the additive dose method. The activation energies and frequency factors obtained using PS and ILD methods are calculated to be 0.72 and 0.72 eV and 8.76 × 10⁵ and 1.44 × 10⁶/s, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Abortiporus biennis tolerance to insoluble metal oxides: oxalate secretion, oxalate oxidase activity, and mycelial morphology

The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu₂O, Al₂O₃, ZnO, CuFe₂O₄Zn, CdO, and MnO₂) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu₂O- and ZnO-amended plates was observed. ZnO, CdO and Cu₂O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu₂O, MnO₂, ZnO and CuFe₂O₄Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu₂O, ZnO and MnO₂. Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number.     

AMMONIUM STIMULATION OF DARK CARBON FIXATION AS AN INDICATOR OF NITROGEN DEFICIENCY IN PHYTOPLANKTON: POTENTIAL ERRORS CAUSED BY AMMONIUM-OXIDIZING BACTERIA1

Stimulation of dark fixation of carbon by NH₄⁺ is often used as an indicator of phytoplankton N deficiency. This assay is based on the influence of available NH₄⁺ on anaplerotic CO₂ fixation by algae. However, carbon fixation by chemoautotrophic NH₄⁺-oxidizing bacteria may also be stimulated by NH₄⁺ enrichment, a process that can mask the algal response in natural communities. NH₄⁺ addition enhanced dark carbon fixation up to 300%, relative to unamended controls, in organisms collected on a 0.7-μm retention filter in oligotrophic Flathead Lake, Montana, but the effect was not detectable in the presence of nitrapyrin, an inhibitor of NH₄⁺-oxidizing bacteria. Dark carbon fixation was enhanced with addition of NH₄⁺ in organisms retained on 2-μm filters (which should allow passage of most bacteria). NH₄⁺ stimulated dark carbon fixation in N-deficient axenic cultures of Chlamydomonas reinhardtii Dang but not in N-replete cultures in both the presence and absence of nitrapyrin. Application of nitrapyrin or size fractionation treatments, to separate the processes of dark carbon fixation by nitrifiers and phytoplankton, may improve the efficacy of assays using NH₄⁺ stimulation of dark carbon fixation to specifically indicate N deficiency in natural algal communities.     

New Phenylpropanoid Glycosides from Illicium majus and Their Radical Scavenging Activities

Chemical investigation of the ethanol extract of the branch and leaves of Illicium majus resulted in the isolation of four new phenylpropanoid glycosides ( 1 – 4 ) and one new phenolic glycoside ( 9 ), along with 13 known ones. Spectroscopic techniques were used to elucidate the structures of the new isolates such as 3-[(2R,3S)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1-benzofuran-5-yl]propyl β-D-glucopyranoside ( 1 ), [(2R,3S)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-2,3-dihydro-1-benzofuran-3-yl]methyl 2-O-α-L-rhamnopyranosyl-β-D-glucopyranoside ( 2 ), [(2R,3S)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-5-(3-hydroxypropyl)-2,3-dihydro-1-benzofuran-3-yl]methyl 2-O-α-L-rhamnopyranosyl-β-D-xylopyranoside ( 3 ), 3-[(2R,3S)-3-({[2-O-(4-O-acetyl-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]oxy}methyl)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydro-1-benzofuran-5-yl]propyl acetate ( 4 ), and 4-(2-hydroxyethyl)phenyl 3-O-β-D-glucopyranosyl-β-D-glucopyranoside ( 9 ). Free radical scavenging activities of the isolates were elucidated through the DPPH assay method. The most active compounds, 1-O-caffeoyl-β-D-glucopyranose ( 17 ) and soulieana acid 1 ( 18 ), exhibited moderate radical scavenging activities (IC₅₀=37.7±4.4 μM and IC₅₀=97.2±3.4 μM, respectively). The antibacterial activities of the isolates against Staphylococcus aureus and Escherichia coli were also assessed, and no activity was shown at the measured concentration (<32 μg/mL).     

Mixed Valence Tin Oxides as Novel van der Waals Materials: Theoretical Predictions and Potential Applications

Van der Waals (vdW) heterostructures, which can be assembled by combining 2D atomic crystals in a precisely chosen sequence, enable a wide range of potential applications in optoelectronics, photovoltaics, and photocatalysis. However, the difficulty of peeling isolated atomic planes and the lattice mismatch between different materials is the main obstacle to hinder vdW materials from more practical applications. In this work, the mixed valence tin oxides, Sn_xO_y (0.5 < x/y < 1), are proposed as a new member of vdW materials and these mixed valence tin oxides show promise to overcome the above‐mentioned obstacle. Density‐functional theory calculations are combined with an evolutionary algorithm to predict the crystal structures of a series of previously reported tin oxides (Sn₂O₃, Sn₃O₄, Sn₄O₅, and Sn₅O₆), unreported compositions (Sn₇O₈, Sn₉O₁₀, and Sn₁₁O₁₂), and a new β ‐ SnO phase. These structures consist of β‐SnO, Sn₂O₃, and Sn₃O₄ monolayers. Their band gaps can be engineered in the 1.56–3.25 eV range by stacking the monolayers appropriately. The band gap depends linearly on the interlayer distance, as understood from interlayer Sn²⁺–Sn²⁺ and intralayer Sn²⁺–O interactions. Sn_xO_y structures exhibit high photoabsorption coefficients and suitable band‐edge positions for photoexcited H₂ evolution; this indicates potential for environmentally benign solar energy conversion in photovoltaic and photocatalytic applications.     

Vibsane‐Type Diterpenoids from Viburnum odoratissimum and Their Cytotoxic and HSP90 Inhibitory Activities

Four new vibsane‐type diterpenoids, vibsanol I ( 1 ), 15‐hydroperoxyvibsanol A ( 2 ), 14‐hydroperoxyvibsanol B ( 3 ), 15‐O‐methylvibsanin U ( 4 ), and a new natural product, 5,6‐dihydrovibsanin B ( 5 ), as well as six known analogues, were isolated from the twigs and leaves of Viburnum odoratissimum. Their structures were elucidated by spectroscopic analyses and chemical derivatization method. All compounds showed different levels of cytotoxicity against five cell lines (HL‐60, A‐549, SMMC‐7721, MCF‐7, and SW480). Remarkably, 14,18‐O‐diacetyl‐15‐O‐methylvibsanin U ( 4a ) showed significant cytotoxicity against HL‐60, A‐549, SMMC‐7721, MCF‐7, and SW480, with IC₅₀ values of 0.15 ± 0.01, 0.69 ± 0.01, 0.41 ± 0.02, 0.75 ± 0.03, and 0.48 ± 0.03 μm , respectively. In addition, vibsanin K ( 10 ) was identified as a HSP90 inhibitor with an IC₅₀ value of 19.16 μm .     

Insecticidal Metabolites from the Rhizomes of Veratrum album against Adults of Colorado Potato Beetle,Leptinotarsa decemlineata

The dried rhizomes of Veratrum album were individually extracted with CHCl₃, acetone, and NH₄OH/benzene to test the toxic effects against the Colorado potato beetle, Leptinotarsa decemlineata, which is an important agricultural pest. Fifteen compounds in various amounts were isolated from the extracts using column and thin‐layer chromatography. The chemical structures of 14 compounds were characterized as octacosan‐1‐ol ( 1 ), β‐sitosterol ( 2 ), stearic acid ( 3 ), diosgenin ( 4 ), resveratrol ( 5 ), wittifuran X ( 6 ), oxyresveratrol ( 7 ), β‐sitosterol 3‐O‐β‐D ‐glucopyranoside ( 8 ), diosgenin 3‐O‐α‐L ‐rhamnopyranosyl‐(1→2)‐β‐D ‐glucopyronoside ( 9 ), oxyresveratrol 3‐O‐β‐D ‐glucopyranoside ( 10 ), jervine ( 11 ), pseudojervine ( 13 ), 5,6‐dihydro‐1‐hydroxyjervine ( 14 ), and saccharose ( 15 ) using UV, IR, MS, ¹H‐ and ¹³C‐NMR, and 2D‐NMR spectroscopic methods. However, the chemical structure of 12 , an oligosaccharide, has not fully been elucidated. Compounds 4, 6, 9 , and 10 were isolated from V. album rhizomes for the first time in the current study. The toxic effects of three extracts (acetone, CHCl₃, and NH₄OH/benzene) and six metabolites, 2, 2 + 4, 5, 7, 8 , and 11 , were evaluated against the Colorado potato beetle. The assay revealed that all three extracts, and compounds 7, 8 , and 11 exhibited potent toxic effects against this pest. This is the first report on the evaluation of the toxic effects of the extracts and secondary metabolites of V. album rhizomes against L. decemlineata. Based on these results, it can be concluded that the extracts can be used as natural insecticides.     

A nanoparticle-based immobilization assay for prion-kinetics study

Magnetic and gold coated magnetic nanoparticles were synthesized by co-precipitation of ferrous and ferric chlorides, and by the micromicelles method, respectively. Synthesized nanoparticles were functionalized to bear carboxyl and amino acid moieties and used as prion protein carriers after carbodiimide activation in the presence of N-hydroxysuccinimide. The binding of human recombinant prion protein (huPrPrec) to the surface of these nanoparticles was confirmed by FTIR and the size and structures of the particles were characterized by transmission electron microscopy. Findings indicate that the rate of prion binding increased only slightly when the concentration of prion in the reaction medium was increased. Rate constants of binding were very similar on Fe₃O₄@Au and Fe₃O₄-LAA when the concentrations of protein were 1, 2, 1.5, 2.25 and 3.57 μg/ml. For a 5 μg/ml concentration of huPrPrec the binding rate constant was higher for the Fe₃O₄-LAA particles. This study paves the way towards the formation of prion protein complexes onto a 3-dimensional structure that could reveal obscure physiological and pathological structure and prion protein kinetics.     

Cytotoxic Activity and Related Mechanisms of Prenylflavonoids Isolated from Mallotus conspurcatus Croizat

Five prenylflavonoids, 6‐prenylnaringenin ( 1 ), 8‐prenylnaringenin ( 2 ), 7‐O‐methyl‐8‐prenylnaringenin ( 3 ), 7‐O‐methyl‐6‐prenylnaringenin ( 4 ), and 4′‐O‐methyl‐6‐prenylnaringenin ( 5 ), were isolated from the traditional herb Mallotus conspurcatus Croizat (Euphorbiaceae). Compounds 1 – 5 revealed cytotoxic activity against cervical cancer (HeLa) cells with IC₅₀ values ranging from 10.08 to 60.16 μm by MTT method, and interestingly, these prenylflavonoids were less toxic to normal HL‐7702 cells. Furthermore, compounds 1 and 5 could inhibit the c‐myc expression and telomerase activity and cause mitochondrial dysfunction. These findings might contribute to a better understanding of the biological activities of prenylflavonoids and lay the foundation for further studies on the cytotoxic activity of natural products isolated from M. conspurcatus.