Fungal profiles in various milk thistle botanicals from US retail.

Milk thistle (MT) dietary supplements are widely consumed due to their possible beneficial effect on liver health. As botanicals, they can be contaminated with a variety of fungi and their secondary metabolites, mycotoxins. This study was conducted in an effort to determine the mycological quality of various MT botanical supplements from the US market. Conventional plating methods were used for the isolation and enumeration of fungi, while conventional microscopy as well as molecular methods were employed for the speciation of the isolated strains. Results showed that a high percentage of the MT samples tested were contaminated with fungi. Total counts ranged between <2.00 and 5.60 log10 colony forming units per gram (cfu/g). MT whole seeds carried the highest fungal levels followed by MT cut herb. No live fungi were recovered from MT seed tea bags, liquid extracts, capsules or soft gels. Potentially toxigenic molds from the Aspergillus sections Flavi and Nigri as well as Eurotium, Penicillium, Fusarium and Alternaria species were isolated from MT supplements. The predominant molds were Eurotia (E. repens, E. amstelodami and E. rubrum), A. flavus, A. tubingensis, A. niger and A. candidus. To our knowledge, this is the first study reporting on fungal contamination profiles of MT botanicals.

Occurrence of aflatoxins in milk thistle herbal supplements.

Milk thistle (MT) dietary supplements are widely consumed due to their possible liver-health-promoting properties. As botanicals they can be contaminated with a variety of fungi and their secondary metabolites, mycotoxins. The aflatoxigenic fungus Aspergillus flavus has been previously isolated from these commodities. Currently, there is no published method for determining aflatoxins (AFs) in MT. Therefore, a liquid chromatography (LC) method validated for aflatoxin analysis in botanicals was evaluated and applied to MT. The method consisted of acetonitrile/water extraction, immunoaffinity column clean-up, LC separation, post-column photochemical reaction derivatisation and fluorescence detection. The average recoveries for AFs added to MT seeds, herb, oil-based liquid extract and alcohol-based liquid extract were 76% or higher. The mean relative standard deviation was <10%. The limit of detection (LOD) was 0.01 µg kg(-1) and the limit of quantification (LOQ) was 0.03 µg kg(-1). The method was used to conduct a small survey. A total of 83 MT samples from the US market were analysed. AFs were detected in 19% of the samples with levels ranging from 0.04 to 2.0 µg kg(-1). Additionally, an aflatoxigenic A. flavus strain from ATTC and an A. parasiticus strain isolated from MT herb powder were found to produce high amounts of aflatoxins (11,200 and 49,100 µg kg(-1), respectively) when cultured in MT seed powder. This is the first study reporting on aflatoxin contamination of MT botanical supplements and identifying methodology for AF analysis of these commodities.

DNA-induced structural changes in the papillomavirus capsid.

Human papillomavirus capsid assembly requires intercapsomeric disulfide bonds between molecules of the major capsid protein L1. Virions isolated from naturally occurring lesions have a higher degree of cross-linking than virus-like particles (VLPs), which have been generated in eukaryotic expression systems. Here we show that DNA encapsidation into VLPs leads to increased cross-linking between L1 molecules comparable to that seen in virions. A higher trypsin resistance, indicating a tighter association of capsomeres through DNA interaction, accompanies this structural change.

A novel retroviral vector that allows the magnetic selection of infected cells.

Retroviral vectors are used widely in research and are also being designed for use in gene therapy trials. In practice, these vectors usually contain a marker gene, which is often a drug selection gene. In this report, a novel retroviral vector has been constructed which contains a gene that allows selection for infected cells by a magnet. This gene is a single-chain antibody (sFv) to a specific hapten molecule 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (phOx). sFv specific for phOx is displayed on the surface of infected cells. This feature allows binding to phOx-BSA coated magnetic beads which are used to isolate the infected cells.

Most retroviral recombinations occur during minus-strand DNA synthesis.

Retroviral RNA molecules are plus, or sense in polarity, equivalent to mRNA. During reverse transcription, the first strand of the DNA molecule synthesized is minus-strand DNA. After the minus strand is polymerized, the plus-strand DNA is synthesized using the minus-strand DNA as the template. In this study, a helper cell line that contains two proviruses with two different mutated gfp genes was constructed. Recombination between the two frameshift mutant genes resulted in a functional gfp. If recombination occurs during minus-strand DNA synthesis, the plus-strand DNA will also contain the functional sequence. After the cell divides, all of its offspring will be green. However, if recombination occurs during plus-strand DNA synthesis, then only the plus-strand DNA will contain the wild-type gfp sequence and the minus-strand DNA will still carry the frameshift mutation. The double-stranded DNA containing this mismatch was subsequently integrated into the host chromosomal DNA of D17 cells, which were unable to repair the majority of mismatches within the retroviral double-strand DNA. After the cell divided, one daughter cell contained the wild-type gfp sequence and the other daughter cell contained the frameshift mutation in the gfp sequence. Under fluorescence microscopy, half the cells in the offspring were green and the other half of the cells were colorless or clear. Thus, we demonstrated that more than 98%, if not all, retroviral recombinations occurred during minus-strand DNA synthesis.

Systematic comparison of a color reporter gene and drug resistance genes for the determination of retroviral titers.

Retroviral vectors usually contain drug resistance genes, which are used to select for infected cells and to determine the viral titers. The viral titer is referred to as colony-forming units (CFUs). Color reporter genes, such as the lacZ gene and the green fluorescent protein gene (gfp), have been widely used as markers in retroviral vectors. In this report, a simple and rapid method for the determination of retroviral titers has been developed. The number of viral particles capable of forming individual green cells per unit volume is defined as marker-forming units (MFUs). The MFUs determined by using gfp as a marker were found to be proportional to the CFUs obtained by using drug selection for five different drug resistance genes. In addition, after adjusting the time factor, the MFUs are higher than CFUs in viruses released from 30 stable helper cell lines. The lower titers determined by CFUs are likely due to the toxicity on transduced cells.

Recombination between two identical sequences within the same retroviral RNA molecule.

As a consequence of being diploid viruses, members of the Retroviridae have a high recombination rate. To measure recombination between two identical sequences within the same RNA molecule per round of retroviral replication cycle, a murine leukemia virus based vector (JZ442 + 3' Hyg) has been constructed. It carries a drug resistance gene, hyg, and a 290-bp repeat sequence of the 3' hyg gene inserted into the 3' untranslated region of the green fluorescent protein gene (gfp). Under fluorescence microscopy, Hygr cells containing the recombinant proviruses were clear, while a green color was observed in the drug-resistant cells carrying the parental proviruses. The rate of recombination was determined by the ratio of the number of clear colonies to the total number of Hygr colonies (green and clear colonies). The rate of recombination was found to be 62% by this method. The intermolecular recombination rate between an infectious virus bearing two copies of the 290-bp segment and a noninfectious chimeric RNA virus containing only a single copy of this sequence was also measured.

Glycine-EDTA treatment to prepare Ko red blood cells: preservation of high-frequency antigens.

Reactivity of high-incidence antigens after glycine-EDTA treatment of red blood cells (RCs) to prepare artificial Ko RBCs was investigated. The treatment had little or no effect on Yta, JMH, Yka, Kna, and McCa. Hy antigen-positive-treated RBCs reacted less well with anti-Hy when compared to nontreated cells. Glycine-EDTA treatment to prepare Ko RBCs offers an advantage over AET treatment because it preserves some high-frequency antigens that AET treatment does not. Immunohematology 1994;10:64-65.

Sodium chloride equivalents, cryoscopic properties, and hemolytic effects of certain medicinals in aqueous solution. III: Supplemental values.

A supplemental table of sodium chloride equivalents and freezing-point depressions at various concentrations for 44 different substances in aqueous solution is presented. Also given in the table is the isosmotic concentration of each material that can form such a solution. The degree of hemolysis of human erythrocytes was determined in 24 different isosmotic solutions, and the data are presented in a table to supplement the previously pub lished values. Eleven isosmotic solutions prevented hemolysis, and 13 others failed to prevent hemolysis.