The Streptomyces coelicolor ssgB gene is required for early stages of sporulation

ssgB was identified as a novel early sporulation gene in Streptomyces coelicolor. An ssgB deletion mutant failed to sporulate, over-produced actinorhodin, and its colonies were significantly larger than those of the parental strain, suggesting an important role for the ssgB gene product in the process of growth cessation prior to sporulation-specific cell division. This places ssgB temporally before the paralogous sporulation gene ssgA. Analysis of ssgB mutant hyphae by electron microscopy and by confocal fluorescence microscopy showed that it was defective in the initiation of sporulation, as no sporulation septa could be identified, and DNA segregation had not yet been initiated in the mutant.     

Identification of key amino acid residues that determine the ability of high risk HPV16-E7 to dysregulate major histocompatibility complex class I expression

High risk human Papillomavirus (HPV) types are the major causative agents of cervical cancer. Reduced expression of major histocompatibility complex class I (MHC I) on HPV-infected cells might be responsible for insufficient T cell response and contribute to HPV-associated malignancy. The viral gene product required for subversion of MHC I synthesis is the E7 oncoprotein. Although it has been suggested that high and low risk HPVs diverge in their ability to dysregulate MHC I expression, it is not known what sequence determinants of HPV-E7 are responsible for this important functional difference. To investigate this, we analyzed the capability to affect MHC I of a set of chimeric E7 variants containing sequence elements from either high risk HPV16 or low risk HPV11. HPV16-E7, but not HPV11-E7, causes significant diminution of mRNA synthesis and surface presentation of MHC I, which depend on histone deacetylase activity. Our experiments demonstrate that the C-terminal region within the zinc finger domain of HPV-E7 is responsible for the contrasting effects of HPV11- and HPV16-E7 on MHC I. By using different loss- and gain-of-function mutants of HPV11- and HPV16-E7, we identify for the first time a residue variation at position 88 that is highly critical for HPV16-E7-mediated suppression of MHC I. Furthermore, our studies suggest that residues at position 78, 80, and 88 build a minimal functional unit within HPV16-E7 required for binding and histone deacetylase recruitment to the MHC I promoter. Taken together, our data provide new insights into how high risk HPV16-E7 dysregulates MHC I for immune evasion.     

A proof of principle experiment for microbeam radiation therapy at the Munich compact light source

Microbeam radiation therapy (MRT), a preclinical form of spatially fractionated radiotherapy, uses an array of microbeams of hard synchrotron X-ray radiation. Recently, compact synchrotron X-ray sources got more attention as they provide essential prerequisites for the translation of MRT into clinics while overcoming the limited access to synchrotron facilities. At the Munich compact light source (MuCLS), one of these novel compact X-ray facilities, a proof of principle experiment was conducted applying MRT to a xenograft tumor mouse model. First, subcutaneous tumors derived from the established squamous carcinoma cell line FaDu were irradiated at a conventional X-ray tube using broadbeam geometry to determine a suitable dose range for the tumor growth delay. For irradiations at the MuCLS, FaDu tumors were irradiated with broadbeam and microbeam irradiation at integral doses of either 3 Gy or 5 Gy and tumor growth delay was measured. Microbeams had a width of 50 µm and a center-to-center distance of 350 µm with peak doses of either 21 Gy or 35 Gy. A dose rate of up to 5 Gy/min was delivered to the tumor. Both doses and modalities delayed the tumor growth compared to a sham-irradiated tumor. The irradiated area and microbeam pattern were verified by staining of the DNA double-strand break marker γH2AX. This study demonstrates for the first time that MRT can be successfully performed in vivo at compact inverse Compton sources.     

Isolation and characterization of Leuconostoc oenos phages from German wines

Summary Thirty-four wines that showed problems during malolactic fermentation were obtained from five different German wineries and were examined for the presence of phages using electron microscopy and the agar spot-test. Phages were discovered in 11 of the wines and host strains were found for ten of these phages. The phages were characterized on the basis of their morphology, host range and protein profiles. Furthermore the ten phages could be divided into four groups by restriction enzyme analysis of the phage DNA. This grouping was consistent with results based on morphology, protein composition and host range analysis. Correspondence to: E. K. Arendt     

Functional expression of human dihydroorotate dehydrogenase (DHODH) in pyr4 mutants of ustilago maydis allows target validation of DHODH inhibitors in vivo

Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.     

Response of HEK293 and CHO cells overexpressing fusiogenic syncytin-1 to mitochondrion-mediated apoptosis induced by antimycin A

Apoptosis is essential for the regulation of cellular homeostasis in the placenta and is also involved in the pathophysiology of pregnancy-related diseases such as pre-eclampsia and intrauterine growth restriction (IUGR). Syncytin-1, a fusiogenic glycoprotein of endogenous-retroviral origin expressed in human trophoblasts, facilitates placental syncytium formation and is found reduced in pre-eclamptic placentas. We focus here on the mitochondrial apoptotic pathway and investigate whether the overexpression of syncytin-1 in HEK293-52 (human embryonic kidney cells) and CHO-52 cells influences the apoptotic response to the mitochondrial inhibitor antimycin A (AA). After the induction of apoptosis by 5 microM AA and incubation for up to 36 h in the absence of serum, the mean apoptotic rate was reduced by 15-30% in syncytin-1 transfected cells compared with mock-transfectants. After 12 h of challenge with AA we found lower cytochrome c levels in the cytoplasmic protein fraction and higher amounts in the mitochondrial fraction in syncytin-1 transfectants compared with mock-transfectants. We observed a decreased Mitotracker Red staining of mitochondria following AA challenge for 24 h in mock-treated CHO cells, in particular, compared with syncytin-1 transfectants. Moreover, we found a reduced activation of caspase 9 in syncytin-1 transfected HEK293-52 cells after 48 h of apoptotic challenge compared to mock-transfectants. However, a high expression of anti-apoptotic Bcl-x(L) was found in both cell types. Using syncytin-1 transfected HEK293-52 cells and CHO-52 cells, we provide initial evidence that syncytin-1 may exert its anti-apoptotic function at the mitochondrial level. A reduced release of cytochrome c followed by a diminished activation of caspase 9 is a possible mechanism.     

Limited Tryptic Proteolysis of the Benzodiazepine Binding Proteins in Different Species Reveals Structural Homologies

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.