Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like the juvenile hormone in some insects. The role of these enzymes in some organisms is still not fully understood.Epoxides are highly reactive valuable intermediates used by the pharmaceutical industry. Enantiopure epoxides are of high value in the production of pharmaceuticals like pain-killers or protease-inhibitors. There are a number of ways to produce enantiopure epoxides, but nowadays an environmentally friendly manner has a high preference. One such environmentally friendly method is the use of the epoxide hydrolases. These enzymes are able to enantioselectively hydrolyze one epoxide-enantiomer to its vicinal diol. By this so-called kinetic resolution, it is possible to obtain both the epoxide and diol enantiopure. Enantiopure diols are also of high value in the fine and pharmaceutical chemistry.The first goal of this project is achieved: the epoxide hydrolase from Rhodotorula glutinis has been isolated and purified. The second goal (optimization of the reaction conditions) has been performed but it is still favorable to further optimize them. Initial experiments of enzyme stability towards temperature and pH has been performed with crude enzyme extracts, not with the purified enzyme. With respect to the third goal (a suitable method for the isolation and separation of epoxide and diol), the use of a recently described membrane reactor is recommended. The performance of this reactor, however, has not been verified for the EH studied.The enzyme is partially characterized. The EH was found to be a membrane associated enzyme. Whether or not it is actually a membrane bound enzyme (and how many times it passes the membrane) is still unknown. The enzyme consists of two (most probably) identical subunits with a molecular mass of 45 kDa. Amino acid analysis revealed that the enzyme belongs to thea/b-hydrolase fold family, because the characteristic catalytic histidine motive (G H F) can be found in the amino acid sequence. The N-terminal sequence, however, could not be detected. The amount of purified enzyme was too low to establish its the three-dimensional structure.With partially purified enzyme sample, the specific activity and enantioselectivity could be enhanced when detergents were added. Non-ionic detergents had the largest positive effects, e.g. the specific activity for 1,2-epoxyhexane and styrene oxide was enhanced three and eight times, respectively. In the same way, the enantioselectivity for 1,2-epoxyhexane and styrene oxide could be enhanced over 10 and nearly 5 times, respectively. In addition, non-ionic detergents had an enzyme stabilizing effect. Anionic detergents had a very clear negative effect: enzyme activities were reduced to 20%.Another method investigated to influence the stability, the activity and the enantioselectivity consists of polymerizing the epoxide hydrolase in a network. The enantioselective conversion of (±)-1,2-epoxyoctane was reversed from a preference for ( R )-1,2-epoxyoctane to ( S )-1,2-epoxyoctane when the enzyme had been imprinted with ( S )-1,2-epoxyoctane prior to co-polymerization. This is the first time that the above mentioned method was successfully performed with a membrane-associated enzyme of thea/b-hydrolase fold family to which EH belongs. The half-life of the immobilized and imprinted biocatalyst was enhanced at least 7-fold. Most remarkable was that washing the immobilized EH with HCl, followed by washing it with buffer, resulted in about 50% of the residual activity, while native EH completely lost its activityThe effect of increasing epoxide amounts (up to 10 mmol per 10 mL of water, leading to phase separations) on both the activity and enantioselectivity has been studied, including the effect of detergents on such two-phase enzymatic conversions. It appeared that cell-free extracts without detergents gave the highest activity at 10 mmol epoxide per 10 mL of water added, without loss of enantioselectivity as compared to 1 mmol epoxide per 10 mL of water emulsions.It is recommended either to use whole cells (overexpressing EH or not) in a bioreactor to produce enantiopure epoxides and diols in large quantities or to use an immobilized-imprinted enzyme polymer for the conversion of smaller quantities of epoxides in an enantioselectivity of your choice. Further investigations to improve both methods (whole cells, the enzyme properties and the immobilization and imprinting procedures) are required to optimize this type of conversions for practical applications

Unraveling prednisolone resistance in pediatric acute lymphoblastic leukemia: towards personalized targeted therapy

__Abstract__ Optimization of treatment protocols and improved risk stratification have enhanced event-free survival rates in pediatric precursor-B acute lymphoblastic leukemia (BCP-ALL) and T-ALL up to 80-90%. Remarkably, these results are obtained without changing the core chemotherapeutic drugs that have been used for decades, including prednisolone, L-asparaginase and vincristine. To cure the remaining 20% of patients and to reduce long-term side effects in survivors, more personalized targeted therapy is warranted. Novel targeted drugs will most likely serve as adjuvants to current chemotherapy regimens. Prednisolone has been shown to be the most pivotal in treating pediatric BCP-ALL, as in vivo and in vitro response to prednisolone is an important predictor for long-term clinical outcome. Hence, to improve clinical outcome drugs need to be found which reverse resistance to prednisolone. My thesis therefore focused on the identification of genes or mechanisms involved in prednisolone resistance, followed by functional studies to determine the prednisolone sensitizing effects and pathobiology of these genes or mechanisms and subsequently studying target specificity and effectiveness of potential new targeted agents. We have identified several prednisolone resistance mechanisms and have shown that several clinically available inhibitors can target these mechanisms leading to increased prednisolone sensitivity in vitro. These inhibitors could be potential therapeutic treatment options to increase survival in BCP-ALL. For one targeted inhibitor specifically that showed terrific results, we are performing a larger follow-up study to hopefully pave the way for clinical trials

Usefulness of multimodal MR imaging in the differential diagnosis of HaNDL and acute ischemic stroke

<p>Abstract</p> <p>Background</p> <p>Syndrome of transient Headache and Neurological Deficits with cerebrospinal fluid Lymphocitosis (HaNDL) is a rare disease which can present with focal neurological deficits and mimic stroke. A neurologist-on-duty faced with a HaNDL patient in the first hours might erroneously decide to use thrombolytic drugs, a non-innocuous treatment which has no therapeutic effect on this syndrome.</p> <p>Case Presentation</p> <p>We present a case where neuroimaging, together with the clinical picture, led to a presumed diagnosis of HaNDL avoiding intravenous thrombolysis.</p> <p>Conclusions</p> <p>This report shows the usefulness of multimodal MR imaging in achieving early diagnosis during an acute neurological attack of HaNDL. Our experience, along with that of others, demonstrates that neuroimaging tests reveal the presence of cerebral hypoperfusion in HaNDL syndrome</p

PRC2 loss induces chemoresistance by repressing apoptosis in T cell acute lymphoblastic leukemia

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming. Here, we report that mitochondria) apoptosis resistance in T cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2). In T-ALL clinical specimens, loss-of-function mutations of PRC2 core components (EZH2, FED, or SUZ12) were associated with mitochondrial apoptosis resistance. In T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action. PRC2 loss induced apoptosis resistance via transcriptional up-regulation of the LIM domain transcription factor CRIP2 and downstream up-regulation of the mitochondrial chaperone TRAP1. These findings demonstrate the importance of mitochondrial apoptotic priming as a prognostic factor in T-ALL and implicate mitochondrial chaperone function as a molecular determinant of chemotherapy response

Pediatric T-cell acute lymphoblastic leukemia

The most common pediatric malignancy is acute lymphoblastic leukemia (ALL), of which T-cell ALL (T-ALL) comprises 10–15% of cases. T-ALL arises in the thymus from an immature thymocyte as a consequence of a stepwise accumulation of genetic and epigenetic aberrations. Crucial biological processes, such as differentiation, self-renewal capacity, proliferation, and apoptosis, are targeted and deranged by several types of neoplasia-associated genetic alteration, for example, translocations, deletions, and mutations of genes that code for proteins involved in signaling transduction, epigenetic regulation, and transcription. Epigenetically, T-ALL is characterized by gene expression changes caused by hypermethylation of tumor suppressor genes, histone modifications, and miRNA and lncRNA abnormalities. Although some genetic and gene expression patterns have been associated with certain clinical features, such as immunophenotypic subtype and outcome, none has of yet generally been implemented in clinical routine for treatment decisions. The recent advent of massive parallel sequencing technologies has dramatically increased our knowledge of the genetic blueprint of T-ALL, revealing numerous fusion genes as well as novel gene mutations. The challenges now are to integrate all genetic and epigenetic data into a coherent understanding of the pathogenesis of T-ALL and to translate the wealth of information gained in the last few years into clinical use in the form of improved risk stratification and targeted therapies. Here, we provide an overview of pediatric T-ALL with an emphasis on the acquired genetic alterations that result in this disease