Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC₅₀). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100–1,000, and the corresponding IC₅₀ values were in the range from 2.2 nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900 nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC₅₀ values were 4.4–10.8 nmol/l for T-2 toxin, 7.5–55.8 mol/l for HT-2 toxin, 600–4,900 nmol/l for DON, 300–2,600 nmol/l for NIV, and 2.2–18.3 nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC₅₀ values ranging from 16.5 nmol/l (T-2 toxin) to 4,500 nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.     

Ochratoxin A in human blood serum – retrospective long-term data

In a long-term study (1990–1997) on ochratoxin A (OTA) in human blood serum, 102 serum samples from 36 persons of the Munich Institute for Hygiene and Technology of Food of Animal Origin were analysed by enzyme immunoassay (EIA), and by high performance liquid chromatography (HPLC) for control. Detection limits were at 50 pg/ml (EIA) and 50–70 pg/ml (HPLC), recoveries were 80–120% (EIA) and 30–60% (LC). OTA was detected in 98% (EIA, 368 ± 217 pg/ml) and 93% (HPLC, 271 ± 170 pg/ml) of samples (maximum 1,290 pg/ml). Using published conversion factors for serum/intake estimates (1.34 or 1.97), the mean daily OTA intake of these 36 persons was 493–725 pg/kg bw. Long-term individual mean OTA levels of nine persons ranged from 162 ± 80 pg/ml to 549 ± 172 pg/ml. Our data were compared with published OTA serum levels (1985–2008) for apparently healthy persons from a total of 30 countries. On a worldwide basis, the mean of means for OTA in human serum was estimated to be 700 pg/ml, corresponding to a mean daily OTA intake of 940–1380 pg/kg bw. This level, which was relatively stable over the last decades, is well below published tolerable daily intake values (14,000–18,000 pg/kg bw).     

Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein

Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to α-1,4-glucans.     

Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.     

The greater seedling high-light tolerance of <Emphasis Type="Italic">Quercus robur</Emphasis> over <Emphasis Type="Italic">Fagus sylvatica </Emphasis>is linked to a greater physiological plasticity

The responses of Quercus robur (oak) and Fagus sylvatica (beech) seedlings to four different light environments (full, 50%, 40% and 15% sunlight) and to a rapid increase in irradiance were explored during the summer, after 2 years of growth in a forest nursery at Nancy (France). Significant differences between the two species were found for most variables. Phenotypic plasticity for morphological variables (root-shoot ratio, leaf size, leaf weight ratio) was higher in beech than in oak, while the reverse was true for anatomical (stomatal density, epidermis thickness, exchange surface area of the palisade parenchyma) and physiological (maximum photosynthetic rate, stomatal conductance, Rubisco activity) variables. Predawn photochemical efficiency (F_v/F_m) was higher in oak than in beech in all light environments except in 15% sunlight. F_v/F_m was significantly lower in 100% sunlight than in the other light environments in beech but not in oak. Maximum photosynthetic rates (A_max) increased with increasing light availability in the two species but they were always higher in oak than in beech. Oak exhibited higher Rubisco activity than beech in full sunlight. The transfer of shade-adapted seedlings to the open caused a decrease of F_v/F_m, which was larger for beech than for oak. Transferred oak but not beech plants recovered gradually to the control F_v/F_m values. The decreased chlorophyll content and the increased non-photochemical quenching observed in high-light beech seedlings were not enough to avoid photoinhibition. The results suggest that a greater tolerance of strong irradiance is linked to an enhanced physiological plasticity (variables related to photosynthesis), while shade tolerance relies on an enhanced plasticity in light-harvesting variables (crown morphology and chlorophyll content).     

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol

Background

Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABA_A receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABA_A receptors and its GABA_A receptor subtype-specificity was established.

Methods

Different recombinant GABA_A receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol.

Results

3 μM 4-O-methylhonokiol is shown here to potentiate responses of the α₁β₂γ₂ GABA_A receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABA_A receptor subtypes to assess GABA_A receptor subunits that are responsible for the potentiating effect.

Conclusion

The much higher potentiation of GABA_A receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol.

General significance

The results point to the use of 4-O-methylhonokiol as a lead for GABA_A receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.     

Use of glacier river-fed estuary channels by juvenile Coho Salmon: transitional or rearing habitats?

Estuaries are among the most productive ecosystems in the world and provide important rearing environments for a variety of fish species. Though generally considered important transitional habitats for smolting salmon, little is known about the role that estuaries serve for rearing and the environmental conditions important for salmon. We illustrate how juvenile coho salmon Oncorhynchus kisutch use a glacial river-fed estuary based on examination of spatial and seasonal variability in patterns of abundance, fish size, age structure, condition, and local habitat use. Fish abundance was greater in deeper channels with cooler and less variable temperatures, and these habitats were consistently occupied throughout the season. Variability in channel depth and water temperature was negatively associated with fish abundance. Fish size was negatively related to site distance from the upper extent of the tidal influence, while fish condition did not relate to channel location within the estuary ecotone. Our work demonstrates the potential this glacially-fed estuary serves as both transitional and rearing habitat for juvenile coho salmon during smolt emigration to the ocean, and patterns of fish distribution within the estuary correspond to environmental conditions.     

Transmission of Methicillin-Resistant Staphylococcus aureus CC398 from Livestock Veterinarians to Their Household Members

There are indications that livestock-associated MRSA CC398 has a reduced human-to-human transmissibility, limiting its impact on public health and justifying modified control measures. This study determined the transmissibility of MRSA CC398 from livestock veterinarians to their household members in the community as compared to MRSA non-CC398 strains. A one-year prospective cohort study was performed to determine the presence of MRSA CC398 in four-monthly nasal and oropharyngeal samples of livestock veterinarians (n  =  137) and their household members (n  =  389). In addition, a cross-sectional survey was performed to detect the presence of MRSA non-CC398 in hospital derived control patients (n  =  20) and their household members (n  =  41). Staphylococcus aureus isolates were genotyped by staphylococcal protein A (spa) typing and multiple-locus variable-number tandem repeat analysis (MLVA). Mean MRSA CC398 prevalence over the study period was 44% (range 41.6–46.0%) in veterinarians and 4.0% (range 2.8–4.7%) in their household members. The MRSA CC398 prevalence in household members of veterinarians was significantly lower than the MRSA non-CC398 prevalence in household members of control patients (PRR 6.0; 95% CI 2.4–15.5), indicating the reduced transmissibility of MRSA CC398. The impact of MRSA CC398 appears to be low at the moment. However, careful monitoring of the human-to-human transmissibility of MRSA CC398 remains important.