Progesterone 5β-reductase of Erysimum crepidifolium: cDNA cloning,expression in Escherichia coli,and reduction of enones with the recombinant protein

Erysimum is a genus of the Brassicaceae family closely related to the genus Arabidopsis. Several Erysimum species accumulate 5β-cardenolides. Progesterone 5β-reductases (P5βRs) first described in Digitalis species are thought to be involved in 5β-cardenolide biosynthesis. P5βRs belong to the dehydrogenase/reductase super-family of proteins. A full length cDNA clone encoding a P5βR was isolated from Erysimum crepidifolium leaves by 5′/3′ RACE-PCR (termed EcP5βR). Subsequently, the P5βR cDNAs of another nine Erysimum species were amplified by RT-PCR using 5′ and 3′ end primers deduced from the EcP5βR cDNA. The EcP5βR cDNA is 1170 bp long and encodes for 389 amino acids. The EcP5βR cDNA was ligated into the vector pQE 30 UA and the recombinant His-tagged protein (termed rEcP5βR) was over-expressed in Escherichia coli and purified by Ni-chelate affinity chromatography. Kinetic constants were determined for progesterone, 2-cyclohexen-1-one, isophorone, and NADPH. The by far highest specificity constant (k_cat K_M^?1) was estimated for 2-cyclohexen-1-one indicating that this monocyclic enone may be more related to the natural substrate of the enzyme than progesterone. The atomic structure of rEcP5βR was modelled using the crystal structure of P5βR from Digitalis lanata 2V6G as the template. All sequence motifs specific for SDRs as well as the NFYYxxED motif typical for P5βR-like enzymes were present and the protein sequence fitted into the template smoothly.     

One- and two-photon induced QD-based energy transfer and the influence of multiple QD excitations.

Due to the increased use of quantum dots (QDs) in diverse laser microscopies, it is interesting to study the excitation pump power and excitation wavelength dependence of QD-based energy transfer (ET) processes. The ET in QD conjugates with phthalocyanines (Pcs) was studied with femtosecond time-resolved pump-probe spectroscopy upon one- and two-photon excitation. At the used excitation wavelengths only the QDs are excited and become the energy donors. Due to the matched spectral overlap of QD photoluminescence and Pc absorption, the ET occurs on a picosecond time scale. The ET process shows strong pump power dependence whereby an increase in excitation power results in multiple QD excitations and in shorter excited state lifetimes on the QDs due to Auger relaxation. As a result, high excitation pump power leads also to an accelerated ET to the acceptor molecules from the initially multiply excited states of the QDs. Excited state quenching studies as function of pump power suggest that ET occurs mainly from the lowest one-exciton state (n = 1) and only to a minor extent from the multiply excited states (n > 1). For the short-lived, multiply excited states the ET competes inefficiently with Auger recombinations and energy transfer efficiencies of phi(ET)(n=1>) approximately 20%, phi(ET)(n=2>) approximately 7%, phi(ET)(n=3>) < or = 2% were obtained. Also after two-photon excitation the ET efficiency is highest from the one-exciton state. The experimentally determined ET efficiencies were compared with theoretical ET efficiencies upon multiple excitations. In both cases the ET efficiency decreases with the increase in excitation pump power.     

A functionally characterized test set of human induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.     

Maturation of spinal motor neurons derived from human embryonic stem cells

Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.     

Antioxidant effects of carotenoids in a model pigment-protein complex

The effect of carotenoids on stability of model photosynthetic pigment-protein complexes subjected to chemical oxidation with hydrogen peroxide or potassium ferricyanide was investigated. The oxidation of carotenoid-less and carotenoid-containing complexes was conducted in the presence or absence of ascorbic acid. The progress of the reactions was monitored by use of absorption and fluorescence spectroscopy. Our results show that carotenoids may significantly enhance the stability of photosynthetic complexes against oxidation and their protective (antioxidant) effect depends on the type of the oxidant.     

Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity

Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.