Penicillium verrucosum in feed of ochratoxin A positive swine herds

Ochratoxin A contamination of cereal feed grain was monitored during October 1989–September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2–45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (2 ng/ml blood) and ochratoxin A negative herds (<2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p < 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p < 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5% ) of the feed samples collected from the ochratoxin A negative herds.     

High-performance liquid chromatographic assay of formycin A in plasma after solid-phase extraction

A new, simple and accurate high-performance liquid chromatography (HPLC) method for the determination of formycin A in plasma is presented. The samples were chromatographed on a LiChrosorb RP-18 column after purification using a Bakerbond SPE column. The mobile phase was methanol–0.067 M phosphate buffer, pH 4.20 (1:4, v/v) containing 0.005 M sodium hexanesulfonate. Azathioprine was applied as an internal standard. UV detection was carried out at 293 nm. The method was tested for linearity (over the range 0.1–9.0 μg/ml). The recovery was 91.89% (mean). The described method has been successfully applied to the quantitative determination of formycin A in plasma and should be useful for clinical and bioavailability investigations.     

A new strain of <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> produces high yield of oligomycin A with potent anti-tumor activity on human cancer cell lines in vitro

A new actinomycete strain, isolated from soil in China, strongly inhibited in vitro proliferation of human hepatoma, chronic myelogenous leukemia, and colonic carcinoma cell lines. The strain, designated L033, was identified as a strain of Streptomyces avermitilis based on cultural property, morphology, carbon source utilization, 16s rRNA gene analysis, and DNA–DNA relatedness studies. The anticancer component from L033 was purified to homogeneity by preparative positive-phase high-performance liquid chromatography and crystallization. Nuclear magnetic resonance and mass spectrometric analysis showed that this compound had the same structure as oligomycin A. Different with other reported naturally occurring strains of S. avermitilis, L033 produced high quantity of oligomycin A (maximal 1,461 μg/ml). Therefore, L033 was considered of great potential as an industrial oligomycin-A-producing strain. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors using liquid chromatography–electrospray mass spectrometry

Employing high-performance liquid chromatography–electrospray mass spectrometry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG-CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the addition of HCl. The reaction product, mevalonolactone, and internal standard, were extracted with ethyl acetate, dissolved in methanol, and analyzed by LC–MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic acid (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m/z 131 and internal standard, β,β-dimethyl-γ-(hydroxymethyl)-γ-butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantitation was approximately 16.3 pg. Extraction recovery was >90%. The relative standard deviations for intra- and inter-day assays were approximately 4.1±2.7 and 9.4±3.4%, respectively. Mevalonolactone was examined over a period of 3 days and found to be stable. Using this assay, lovastatin and mevastatin inhibited HMG-CoA reductase activity with IC₅₀ values 0.24±0.02 and 2.16±0.31 μM, respectively. These methods offer some advantages over those reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum cholesterol or alter cell growth and differentiation.     

Concentration-dependent inducing activity of activin A

Summary Human recombinant activin A, which is identical with erythroid differentiation factor (EDF), was tested for its mesoderm-inducing activity in concentrations from 0.3–50 ng/ml, using ectoderm of Xenopus late blastula (Stage 9) as the responding tissue. At a low concentration of activin A, blood-like cells, mesenchyme, and coelomic epithelium were induced; at a moderate concentration muscle and neural tissue, and at a high concentration notochord. Activin A thus induced all mesodermal tissues in a dose-dependent manner, such that a low dose induced ventral structures and a high dose induced dorsal structures. Activin may act as an intrinsic inducing molecule responsible for establishing the dorso-ventral axis in early Xenopus development. Offprint requests to: M. Asashima     

A New Long‐Chain Alkene and Antituberculosis Constituents from the Leaves of Pourthiaea lucida

A new long‐chain alkene, dotriacont‐1‐ene ( 1 ), was isolated from the leaves of Pourthiaea lucida, together with twelve known compounds. The structure of this new compound was determined by NMR and mass‐spectrometric analyses. Among the isolated compounds, α‐tocospiro A ( 2 ), α‐tocopheryl quinone ( 4 ), and (E)‐phytol ( 5 ) exhibited antituberculosis activities (MICs ≤30 μg/ml) against Mycobacterium tuberculosis H₃₇Rv in vitro.     

Determination of Guan-Fu Base A, a new anti-arrhythmic, in human plasma by gas chromatography and electron–capture detection

A gas chromatographic–electron capture detection (GC–ECD) method has been developed for determining Guan-Fu Base A (GFA), an experimental anti-arrhythmic, in human plasma. The method was based on one-step liquid–liquid extraction with toluene and chemical derivatization with pentafluoropropionic anhydride followed by GC–ECD. The derivatives of GFA and metoprolol (Met, internal standard) were confirmed by gas chromatography–mass spectrometry (GC–MS) to be dipentafluoropropionyl-GFA and dipentafluoropropionyl-Met. The method was linear over the concentration ranges of 0.1–20.0 and 1.0–30.0 μg/ml with the detection limit of 0.05 μg/ml at S/N=5. The intra- and inter-assay precisions were less than 6 and 10%, and accuracy 99.70±3.30 and 97.60±5.99%, respectively. The absolute recoveries were 81.88, 77.35, 80.79 and 83.85% for GFA at concentrations of 0.5, 1.0, 5.0 and 14.0 μg/ml and 88.24% for Met at 3.0 μg/ml, respectively.     

Morphological Alterations of Saccharomyces cerevisiae Induced by Benanomicin A,an Antifungal Antibiotic with Mannan Affinity

The effects of benanomicin A, a mannose-binding antifungal antibiotic, on yeast cells of Saccharomyces cerevisiae were studied by electron microscopy. Cytological studies using vital stain with methylene blue demonstrated that benanomicin A at 20 and 80 μg/ml killed buds in preference to parent cells. In confirmation, examination by TEM revealed that benanomicin A at 80 μg/ml damaged buds more severely than parent cells. The major effect on the ultrastructure was characterized by severe damage to the cell membrane. In addition, it caused expansion and vacuolation of the endoplasmic reticulum (ER), and partial fragmentation and disappearance of nuclear membranes. The membrane-disruptive activity of benanomicin A may be closely associated with its membrane affinity.     

Improvement of glidobactin A production byPolyangium brachysporum

Summary Glidobactins A, B and C are lipopeptide antitumor antibiotics produced by the gliding bacteriumPolyangium brachysporum sp. nov. No. K481-B101. The production of glidobactin A was examined in shake flasks and laboratory fermentors. Medium screening and optimization led to approximately five fold increases in glidobactin A titers in shake flasks and a ten fold increase in titers in 40-1 batch fermentations. Utilization of a stepped glucose feeding protocol resulted in glidobactin A titers of 1860 g/ml after 144 h of fermentation.     

Biomonitoring of ochratoxin A in grain workers

Handling agricultural commodities such as grain can result in an inhalation of mycotoxin-containing dusts. Ochratoxin A (OTA) is particularly well suited for biomonitoring studies due to its long half-life in blood, and served as a marker toxin to investigate whether or not exposure to dusts in occupational contexts may result in elevated OTA blood serum levels. OTA analysis was performed for blood samples (n=61) obtained from a cohort of male workers employed at granaries of several grain handling companies in Germany. OTA was analyzed in plasma extracts by HPLC with fluorimetric detection; calibration curves were run for each batch of samples collected between July 2005 and March 2006, and the level of detection was 0.05 ng/ml plasma. The OTA plasma levels of the 61 grain workers ranged between 0.07 ng/ml and 0.75 ng/ml. The mean (0.28±0.13 ng/ml) and median (0.26 ng/ml) OTA value for this cohort was similar to average values previously reported for the German population. Our results gave no indication that OTA in excess of those originating from typical dietary sources was ingested by these workers. Although measurable OTA concentrations have been found in dust samples collected at the corresponding workplaces (Mayeret al, this issue), the biomonitoring data do not provide evidence for a significant inhalatory burden of OTA in grain workers. Since deoxynivalenol and zearalenone were also detected in the dust samples in concentrations much higher than that of OTA, additional research should try to assess the potential relevance of an inhalation exposure to these mycotoxins. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

A strategy to obtain axenic cultures of <Emphasis Type="Italic">Arthrospira</Emphasis> spp. cyanobacteria

A strategy to obtain axenic cultures of the cyanobacterium Arthrospira sp. (‘platensis’) Lefevre 1963/M-132-1 strain, consisting of a series of physical and chemical procedures, and the application of an optimized pool of antibiotics, is described in this paper. This strategy, which is an inexpensive and fast way to obtain axenic cultures, can be applied to Arthrospira spp. from culture collections or samples from their natural habitats to eliminate a wide spectrum of contaminants. A high alkaline treatment (pH 12, using KOH) of 72 h is a determinant initial procedure applied to eliminate protozoa and Microcystis sp. Bacteria were eliminated by an optimal antibiotic pool treatment, and Chroococcus sp. residuals were discarded by serial dilution. Optimal concentrations of the antibiotics composing the pool were obtained by a 2⁴ factorial central composite rotatable design (CCRD) and Response Surface Methodology (RSM), resulting in: ampicillin 61.6 μg/ml, penicillin 85.8 μg/ml, cefoxitin 76.9 μg/ml, and meropenem 38.9 μg/ml. The results also indicate that cefoxitin was the most effective antibiotic of this pool. After obtaining the axenic culture, identification of Lefevre 1963/M-132-1 strain was performed using amplification and sequencing of the ITS region (including part of 16S rRNA, tRNA Ile, ITS, tRNA Ala and part of 23S rRNA region) and fatty acid composition data. Data base comparison revealed that Lefevre strain is closely related to A. platensis species (99% identity), while fatty acid composition data suggested A. maxima. These seemingly contradictory results are discussed.     

Agglutination of Trypanosoma cruzi by Concanavalin A*

Epimastigotes of Trypanosoma cruzi obtained in culture agglutinate readily with low concentrations of concanavalin A (Con A). Agglutination was linear with time up to 10 min providing that the initial cell density was greater than 1 × 10⁸ cells/ml. Under these conditions, the percentage agglutination was dependent on the Con A concentration. Agglutination was inhibited by α-methyl D-mannoside, α-D-mannose, and α-D-glucose. Pretreatment of cells with trypsin had no effect on the epimastigote agglutinations. Blood forms (trypomastigotes) of T. cruzi did not agglutinate even in the presence of 100 times more Con A. Results suggest differences in membrane structure between blood forms and cultured epimastigotes of T. cruzi. These membrane differences might be related to the different pathogenic properties of both cell forms of T. cruzi.     

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.     

Annual variations in plasma retinol and alpha-tocopherol levels in gentoo and rockhopper penguins

Blood samples were taken throughout the year from captive gentoo Pygoscelis papua (n = 5) and rockhopper Eudyptes crestatus (n = 10) penguins to measure seasonal variations in retinol and alpha-tocopherol levels. Retinol levels ranged from .58 μg/ml to 1.09 μg/ml and alpha-tocopherol levels from 30.8 μg/ml to 50.7 μg/ml for the gentoo penguins. The rockhopper penguins' retinol levels ranged from .63 μg/ml to 1.14 μg/ml and alpha-tocopherol from 22.3 μg/ml to 40.8 μg/ml. Changes in body mass were used as an indicator of the start and duration of feather moult. Food consumption was recorded daily for the year, and the vitamin A and E intake was calculated. Blood vitamin levels of penguins on a supplemented diet were similar to those in the wild. © 1993 Wiley-Liss, Inc.     

A new strain of <Emphasis Type="Italic">Arthrinium phaeospermum</Emphasis> isolated from <Emphasis Type="Italic">Carex kobomugi</Emphasis> Ohwi is capable of gibberellin production

Plant growth-promoting endophytic fungi with gibberellin-producing ability were isolated from the roots of Carex kobomugi Ohwi, a common sand-dune plant, and bioassayed for plant growth-promotion. A new strain, Arthrinium phaeospermum KACC43901, promoted growth of waito-c rice and Atriplex gemelinii. Analysis of its culture filtrate showed the presence of bioactive GA₁ (0.5 ng/ml), GA₃ (8.8 ng/ml), GA₄ (4.7 ng/ml) and GA₇ (2.2 ng/ml) along with physiologically inactive GA₅ (0.4 ng/ml), GA₉ (0.6 ng/ml), GA₁₂ (0.4 ng/ml), GA₁₅ (0.4 ng/ml), GA₁₉ (0.9 ng/ml) and GA₂₄ (1.8 ng/ml). The fungal isolate was identified through sequence homology and phylogenetic analysis of 18S rDNA (internal transcribed region). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antioxidant properties of dihydroherbimycin A from a newly isolated <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

During antioxidant screening using 1,1-diphenyl-picrylhydrazyl (DPPH) and a lipid peroxidation assay, a streptomycete strain was found to produce herbimycin A and dihydroherbimycin A as antioxidants in the culture filtrate. These molecules were identified by using spectral analyses, including infrared, ultraviolet, mass spectrum, and nuclear magnetic resonance assays. In the DPPH radical-scavenging assay, dihydroherbimycin A exhibited more potent antioxidant activity (IC₅₀, 1.3 μM) than α-tocopherol (IC₅₀, 2.7 μM) that was used as a reference compound. In the lipid peroxidation assay, both herbimycin A and dihydroherbimycin A demonstrated antioxidant activities of 61% and 72%, respectively, at 100 μg/ml, while α-tocopherol exhibited an activity of 93% at the same concentration. Therefore, dihydroherbimycin A might have the potential to be developed into a new therapeutic agent.     

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.