Production of Vero cytotoxin by strains of Escherichia coli as related to the availability of iron

Abstract Vero cytotoxin (VT) producing strains of Escherichia coli (VTEC), including isolates from cases of haemolytic uraemic syndrome and infantile diarrhoea, were used to determine the effect of iron availability on the production of intra- and extracellular VT, with particular interest in elevating toxin production by low-level toxin producing VTEC. Culturing bacteria under iron restriction resulted in growth retardation and a decrease in the production of VT. For the routine detection of both high- and low-level VT-producing E. coli , there was no advantage to be gained by growing bacteria under iron restriction or using disrupted bacterial cell preparations; on the contrary, testing culture supernatants from bacteria grown in iron-replete media for approximately 14 h proved to be the most sensitive and accurate method for detecting VT and the resultant identification of VTEC.     

Biodegradation of phenanthrene by Arthrobacter polychromogenes isolated from a contaminated soil

Summary Degradation of phenanthrene by Arthrobacter polychromogenes isolated from a contaminated soil was investigated. In experiments in which [9-¹⁴C]-phenanthrene was incubated with cultures of A. polychromogenes containing 150 mg phenanthrene/l it was shown that after 26 h of incubation 47.7% of the recovered radiolabelled carbon originally present was metabolized to ¹⁴CO₂, 47.8% was recovered from the aqueous fraction, and 4.5% remained in the dichloromethane fraction. Increasing phenanthrene concentration in the culture medium resulted in improved growth and degradation rates, probably due to the higher amount of phenanthrene crystals in the medium. Shifting the temperature from 30°C to 35°C did not influence phenanthrene degradation significantly but inhibited cell division of A. polychromogenes. Medium supplementation with glucose led to stimulation of phenanthrene degradation at low amounts of glucose (0.45 g/l) whereas at higher concentrations (3 g/l) phenanthrene mineralization decreased.Professor Dr. D. Behrens dedicated to his 65th birthdayOffprint requests to: H.-J. Rehm     

Nanoparticulate transport of oximes over an in vitro blood-brain barrier model

Background

Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB.

Methodology/Principal Findings

In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes.

Conclusions/Significance

With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulations are promising formulations for the treatment of the peripheral and the CNS after OP poisoning.     

Identification of Coffee Genes Expressed During Systemic Acquired Resistance and Incompatible Interaction with Hemileia vastatrix

Coffee genes associated with systemic acquired resistance (SAR) and incompatible reaction against coffee leaf rust inoculation were identified by suppression subtractive hybridization. Analysis of 384 clones of each of the subtracted cDNA libraries identified genes involved in oxidative burst/apoptosis/hypersensitive response, synthesis of antimicrobial proteins, synthesis and transport of antimicrobial metabolites, signal perception and transduction, metabolism of lipids, regulated protein degradation and cell maintenance and development. Induction of distinct sets of genes in the two resistance responses was observed. A wide range of genes involved in defence responses described in other plant species was also found in coffee plants. Semi-quantitative and quantitative RT-PCR analysis of seven selected genes showed differences in their expression profile within 72 h after treatment. Full-length cDNA sequences of two β-1,3-glucanases, one induced during SAR and the other in the incompatible reaction, were obtained by 5' and 3' RACE and the sequence data suggest different properties and cellular localization of the encoded proteins.     

Radiobiology of ultrasoft X rays. III. Normal human fibroblasts and the significance of terminal track structure in cell inactivation

Ultrasoft characteristic X rays from carbon (0.28 keV) are severely attenuated as they pass through biological material, causing a nonuniform distribution of dose to cell nuclei. Complications of studying ultrasoft X rays can be minimized in this context by using cells with very thin cytoplasm and nuclei (e.g., less than the attenuation length of the X rays), and which exhibit a more nearly exponential dose response to cell killing, such as normal human fibroblasts compared with V79 cells. Using this cell system, we report the relative biological effectiveness (RBE) of A1-K and C-K X rays to be near unity. Previous studies of cell inactivation by characteristic carbon X rays gave RBEs of 3 to 4, supporting the idea that localized energy depositions from secondary electrons and primary track ends represent the principal mode of biological action for other low-LET radiations. In part, the reported high RBEs result from the use of mean dose to describe energy deposited within the cell nuclei by these poorly penetrating radiations. Implicit in the use of mean dose is that cellular damage varies linearly with dose within a critical target(s), an assumption that is of questionable validity for cells that exhibit pronounced curvilinear dose responses. The simplest interpretation of the present findings is that most energy depositions caused by track-end effects are not necessarily more damaging than the sparsely ionizing component.     

A phosphorylation code of the Aspergillus nidulans global regulator VelvetA (VeA) determines specific functions

The velvet protein VeA is a global fungal regulator for morphogenetic pathways as well as for the control of secondary metabolism. It is found exclusively in filamentous fungi, where it fulfills conserved, but also unique functions in different species. The involvement of VeA in various morphogenetic and metabolic pathways is probably due to spatially and timely controlled specific protein–protein interactions with other regulators such as phytochrome (FphA) or velvet‐like proteins (VelB). Here we present evidence that Aspergillus nidulans VeA is a multi‐phosphorylated protein and hypothesize that at least four specific amino acids (T167, T170, S183 and Y254) undergo reversible phosphorylation to trigger development and sterigmatocystin biosynthesis. Double mutation of T167 to valine and T170 to glutamic acid exerted the largest effects with regards to sexual development and veA gene expression. In comparison with wild‐type VeA, which shuttles out of the nuclei after illumination this VeA variant showed stronger nuclear accumulation than the wild type, independent of the light conditions. The interaction between VeA and VelB or FphA, respectively, was affected in the T167V‐T170E mutant. Our results suggest complex regulation of the phosphorylation status of the VeA protein.     

In toto differentiation of human amniotic membrane towards the Schwann cell lineage

Human amniotic membrane (hAM) is a tissue containing cells with proven stem cell properties. In its decellularized form it has been successfully applied as nerve conduit biomaterial to improve peripheral nerve regeneration in injury models. We hypothesize that viable hAM without prior cell isolation can be differentiated towards the Schwann cell lineage to generate a possible alternative to commonly applied tissue engineering materials for nerve regeneration. For in vitro Schwann cell differentiation, biopsies of hAM of 8 mm diameter were incubated with a sequential order of neuronal induction and growth factors for 21 days and characterized for cellular viability and the typical glial markers glial fibrillary acidic protein (GFAP), S100β, p75 and neurotrophic tyrosine kinase receptor (NTRK) using immunohistology. The secretion of the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) was quantified by ELISA. The hAM maintained high viability, especially under differentiation conditions (90.2 % ± 41.6 day 14; 80.0 % ± 44.5 day 21 compared to day 0). Both, BDNF and GDNF secretion was up-regulated upon differentiation. The fresh membrane stained positive for GFAP and p75 and NTRK, which was strongly increased after culture in differentiation conditions. Especially the epithelial layer within the membrane exhibited a change in morphology upon differentiation forming a multi-layered epithelium with intense accumulations of the marker proteins. However, S100β was expressed at equal levels and equal distribution in fresh and cultured hAM conditions. Viable hAM may be a promising alternative to present formulations used for peripheral nerve regeneration.