Importance of the cytochrome P450 monooxygenase CYP52 family for the sophorolipid-producing yeast Candida bombicola

Three cytochrome P450 monooxygenases belonging to the CYP52 family were isolated from the genome of the sophorolipid-producing yeast Candida bombicola using degenerate PCR and genomic walking. One gene displayed high identity with the CYP52E members and was classified into this group ( CYP52E3 ), whereas the other genes belonged to new groups: CYP52M and CYP52N. CYP52E3 and CYP52N1 turned out to be of no relevance for sophorolipid production, but show clear upregulation when the yeast cells are grown on alkanes as the sole carbon source. On the other hand, CYP52M1 is clearly upregulated during sophorolipid synthesis and very likely takes part in sophorolipid formation.     

Promoter knock-in: a novel rational method for the fine tuning of genes

Background  

Metabolic engineering aims at channeling the metabolic fluxes towards a desired compound. An important strategy to achieve this is the modification of the expression level of specific genes. Several methods for the modification or the replacement of promoters have been proposed, but most of them involve time-consuming screening steps. We describe here a novel optimized method for the insertion of constitutive promoters (referred to as "promoter knock-in") whose strength can be compared with the native promoter by applying a promoter strength predictive (PSP) model.     

Fenofibrate Increases Very Low Density Lipoprotein Triglyceride Production Despite Reducing Plasma Triglyceride Levels in APOE*3-Leiden.CETP Mice

The peroxisome proliferator-activated receptor alpha (PPARα) activator fenofibrate efficiently decreases plasma triglycerides (TG), which is generally attributed to enhanced very low density lipoprotein (VLDL)-TG clearance and decreased VLDL-TG production. However, because data on the effect of fenofibrate on VLDL production are controversial, we aimed to investigate in (more) detail the mechanism underlying the TG-lowering effect by studying VLDL-TG production and clearance using APOE*3-Leiden.CETP mice, a unique mouse model for human-like lipoprotein metabolism. Male mice were fed a Western-type diet for 4 weeks, followed by the same diet without or with fenofibrate (30 mg/kg bodyweight/day) for 4 weeks. Fenofibrate strongly lowered plasma cholesterol (−38%) and TG (−60%) caused by reduction of VLDL. Fenofibrate markedly accelerated VLDL-TG clearance, as judged from a reduced plasma half-life of glycerol tri[³H]oleate-labeled VLDL-like emulsion particles (−68%). This was associated with an increased post-heparin lipoprotein lipase (LPL) activity (+110%) and an increased uptake of VLDL-derived fatty acids by skeletal muscle, white adipose tissue, and liver. Concomitantly, fenofibrate markedly increased the VLDL-TG production rate (+73%) but not the VLDL-apolipoprotein B (apoB) production rate. Kinetic studies using [³H]palmitic acid showed that fenofibrate increased VLDL-TG production by equally increasing incorporation of re-esterified plasma fatty acids and liver TG into VLDL, which was supported by hepatic gene expression profiling data. We conclude that fenofibrate decreases plasma TG by enhancing LPL-mediated VLDL-TG clearance, which results in a compensatory increase in VLDL-TG production by the liver.     

Proteomic characterization of Her2/neu-overexpressing breast cancer cells

The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients.     

Identification of Two Critically Deleted Regions within Chromosome Segment 7q35-q36 in EVI1 Deregulated Myeloid Leukemia Cell Lines

Chromosomal rearrangements involving the EVI1 proto-oncogene are a recurrent finding in myeloid leukemias and are indicative of a poor prognosis. Rearrangements of the EVI1 locus are often associated with monosomy 7 or cytogenetic detectable deletions of part of 7q. As EVI1 overexpression alone is not sufficient to induce leukemia, loss of a 7q tumour suppressor gene might be a required cooperating event. To test this hypothesis, we performed high-resolution array comparative genomic hybridization analysis of twelve EVI1 overexpressing patients and three EVI1 deregulated cell lines to search for 7q submicroscopic deletions. This analysis lead to the delineation of two critical regions, one of 0.39 Mb on 7q35 containing the CNTNAP2 gene and one of 1.33 Mb on chromosome bands 7q35–q36 comprising nine genes in EVI1 deregulated cell lines. These findings open the way to further studies aimed at identifying the culprit EVI1 implicated tumour suppressor genes on 7q.     

Oxygen depletion during and after mTHPC-mediated photodynamic therapy in RIF1 and H-MESO1 tumors

During photodynamic therapy (PDT), low oxygenation levels, induced both by oxygen consumption and by vascular occlusion, can lead to an inefficient photochemical reaction that may compromise the efficacy of PDT. In the present studies, tumor oxygenation was measured before, during and after meta-tetrahydroxyphenylchlorin (mTHPC)-mediated PDT of murine RIF1 tumors and human mesothelioma xenografts (H-MESO1). Tumor pO2 was measured in real time with Eppendorf polarography, and the extent of relative hypoxia at specific times was measured by immunohistochemical staining. Significant decreases in median pO2 values, as well as an increase in the number of values below 2.5 mmHg, were seen during and after PDT in RIF1 tumors, although there was a large intertumoral variation. Tumor pO2 values did not change significantly in H-MESO1 tumors. Staining with antibodies against the hypoxia marker EF3 showed significant increases in relative hypoxia after PDT in both tumor types compared with separate groups of untreated controls. Our results are consistent with PDT-induced oxygen depletion (reduced pO2) leading to an increase in relative hypoxia in RIF1 tumors. Extensive necrosis in the H-MESO1 tumors may have prevented the detection of PDT-induced hypoxia using the Eppendorf polarographic needle, whereas immunohistochemistry did reveal increases in relative hypoxia.     

Regeneration of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) by a cyclic organogenic system

In a five-step procedure, plants were regenerated from meristematic tissue initiated from nodal tissue in four pea cultivars (Espace, Classic, Solara, and Puget). In step 1, stem tissue with one node (1-cm size) was subcultured on medium containing thidiazuron. As a result multiple shoots were produced, appearing normal or swollen at their bases. The multiple shoots were subcultured in the same medium, resulting in the formation of a green hyperhydric tissue in the swollen bases of the multiple shoots, which is fully covered with small buds [bud-containing tissue (BCT)]. In step 2, BCT fragments were isolated and subcultured in the same medium and, as a result, they were able to reproduce themselves in a cyclic fashion. In step 3, subculture of BCT on medium supplemented with a combination of gibberelic acid, 6-benzyladenine and -naphthalene acetic acid (NAA), resulted in the formation of shoots, which were rooted in step 4 on medium supplemented with 0.5 mg/l NAA, indole-3-acetic acid (IAA) or indole-3-butyric acid. In step 5, in vitro plants were transferred to the greenhouse for acclimatisation and further development. The four varieties tested were all able to produce meristematic tissue, suggesting that its production is genotype independent.     

Transfer of central nervous system autoantigens and presentation in secondary lymphoid organs

Dendritic cells are thought to regulate tolerance induction vs immunization by transferring Ags and peripheral signals to draining lymph nodes (LN). However, whether myelin Ag transfer and presentation in LN occurs during demyelinating brain disease is unknown. In this study, we demonstrate redistribution of autoantigens from brain lesions to cervical LN in monkey experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Immunohistochemical analysis revealed significantly more cells containing myelin Ags in cervical LN of monkeys with EAE compared with those of healthy control monkeys. Myelin Ags were observed in cells expressing dendritic cell/macrophage-specific markers, MHC class II, and costimulatory molecules. Moreover, these cells were directly juxtaposed to T cells, suggesting that cognate interactions between myelin-containing APC and T cells are taking place in brain-draining LN. Indeed, myelin Ag-reactive T cells were observed in cervical LN from marmosets and rhesus monkeys. Importantly, these findings were paralleled by our findings in human tissue. We observed significantly more myelin Ag-containing cells in LN of individuals with MS compared with those of control individuals. These cells expressed APC markers, as observed in marmosets and rhesus monkeys. These findings suggest that during MS and EAE, modulation of T cell reactivity against brain-derived Ags also takes place in cervical LN and not necessarily inside the brain. A major implication is that novel therapeutic strategies may be targeted to peripheral events, thereby circumventing the blood-brain barrier.