Crystallization of the regulatory and effector domains of the key sporulation response regulator Spo0A

The key response-regulator gene of sporulation, spo0A, has been cloned from Bacillus stearothermophilus and the encoded protein purified. The DNA-binding and phospho-acceptor domains of Spo0A have been prepared by tryptic digestion of the intact protein and subsequently crystallized in forms suitable for X-ray crystallographic studies. The DNA-binding domain has been crystallized in two forms, one of which diffracts X-rays to beyond 2.5 Angstrom spacing. The crystals of the phospho-acceptor domain diffract X-rays beyond 2.0 Angstrom spacing using synchrotron radiation

Effects of erythropoietin in murine-induced pluripotent cell-derived panneural progenitor cells

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1–3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of β-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis

Light induced single molecule frequency shift

Alight induced frequency shift of the 0-0 line was measured in two-photon excitation spectra of single diphenyloctatetraene molecules doped in a crystal matrix. The shifts were proportional to the laser power with a slope of about 600 MHz/W when the laser beam of about 300 mW power was focused to a diameter of 2 mu m. Significantly, the observed line broadenings were an order of magnitude smaller than the shifts. The effect is ascribed mainly to a ''fast'' energy exchange between a local vibration and thermal phonons created by the third harmonic C-H band absorption in the matrix, and partially to an ac Stark shift

Absence of system xc⁻ on immune cells invading the central nervous system alleviates experimental autoimmune encephalitis

Background: Multiple sclerosis (MS) is an autoimmune demyelinating disease that affects the central nervous system (CNS), leading to neurodegeneration and chronic disability. Accumulating evidence points to a key role for neuroinflammation, oxidative stress, and excitotoxicity in this degenerative process. System x(c)- or the cystine/glutamate antiporter could tie these pathological mechanisms together: its activity is enhanced by reactive oxygen species and inflammatory stimuli, and its enhancement might lead to the release of toxic amounts of glutamate, thereby triggering excitotoxicity and neurodegeneration. Methods: Semi-quantitative Western blotting served to study protein expression of xCT, the specific subunit of system x(c)-, as well as of regulators of xCT transcription, in the normal appearing white matter (NAWM) of MS patients and in the CNS and spleen of mice exposed to experimental autoimmune encephalomyelitis (EAE), an accepted mouse model of MS. We next compared the clinical course of the EAE disease, the extent of demyelination, the infiltration of immune cells and microglial activation in xCT-knockout (xCT(-/-)) mice and irradiated mice reconstituted in xCT(-/-) bone marrow (BM), to their proper wild type (xCT(+/+)) controls. Results: xCT protein expression levels were upregulated in the NAWM of MS patients and in the brain, spinal cord, and spleen of EAE mice. The pathways involved in this upregulation in NAWM of MS patients remain unresolved. Compared to xCT(+/+) mice, xCT(-/-) mice were equally susceptible to EAE, whereas mice transplanted with xCT(-/-) BM, and as such only exhibiting loss of xCT in their immune cells, were less susceptible to EAE. In none of the above-described conditions, demyelination, microglial activation, or infiltration of immune cells were affected. Conclusions: Our findings demonstrate enhancement of xCT protein expression in MS pathology and suggest that system x(c)- on immune cells invading the CNS participates to EAE. Since a total loss of system x(c)- had no net beneficial effects, these results have important implications for targeting system x(c)- for treatment of MS

Regioselective glucosidation of trans-resveratrol in Escherichia coli expressing glucosyltransferase from Phytolacca americana

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue

Towards broad spectrum activity-based glycosidase probes: synthesis and evaluation of deoxygenated cyclophellitol aziridines

Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of [small beta]-glucosidases and [small beta]-galactosidases

Structural and functional insight into human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) removes O-linked N-acetylglucosamine (O-GlcNAc) from a myriad of nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments, we determined the three-dimensional structure of human OGA, revealing an unusual helix-exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors that have clinical value

Molecular identification of 1-Cys peroxiredoxin and anthocyanidin/flavonol 3-O-galactosyltransferase from proanthocyanidin-rich young fruits of persimmon (Diospyros kaki Thunb.)

Fruits of persimmon (Diospyros kaki Thunb.) accumulate large amounts of proanthocyanidins (PAs) in the early stages of development. Astringent (A)-type fruits remain rich in soluble PAs even after they reach full-mature stage, whereas non-astringent (NA)-type fruits lose these compounds before full maturation. As a first step to elucidate the mechanism of PA accumulation in this non-model species, we used suppression subtractive hybridization to identify transcripts accumulating differently in young fruits of A- and NA-type. Interestingly, only a few clones involved in PA biosynthesis were identified in A–NA libraries. Represented by multiple clones were those encoding a novel 1-Cys peroxiredoxin and a new member of family 1 glycosyltransferases. Quantitative RT-PCR analyses confirmed correlation of the amount of PAs and accumulation of transcripts encoding these proteins in young persimmon fruits. Furthermore, the new family 1 glycosyltransferase was produced in Escherichia coli and shown to efficiently catalyze galactosylation at 3-hydroxyl groups of several anthocyanidins and flavonols. These findings suggest a complex mechanism of PA accumulation in persimmon fruits

A Cross-Study Transcriptional Analysis of Parkinson's Disease

The study of Parkinson's disease (PD), like other complex neurodegenerative disorders, is limited by access to brain tissue from patients with a confirmed diagnosis. Alternatively the study of peripheral tissues may offer some insight into the molecular basis of disease susceptibility and progression, but this approach still relies on brain tissue to benchmark relevant molecular changes against. Several studies have reported whole-genome expression profiling in post-mortem brain but reported concordance between these analyses is lacking. Here we apply a standardised pathway analysis to seven independent case-control studies, and demonstrate increased concordance between data sets. Moreover data convergence increased when the analysis was limited to the five substantia nigra (SN) data sets; this highlighted the down regulation of dopamine receptor signaling and insulin-like growth factor 1 (IGF1) signaling pathways. We also show that case-control comparisons of affected post mortem brain tissue are more likely to reflect terminal cytoarchitectural differences rather than primary pathogenic mechanisms. The implementation of a correction factor for dopaminergic neuronal loss predictably resulted in the loss of significance of the dopamine signaling pathway while axon guidance pathways increased in significance. Interestingly the IGF1 signaling pathway was also over-represented when data from non-SN areas, unaffected or only terminally affected in PD, were considered. Our findings suggest that there is greater concordance in PD whole-genome expression profiling when standardised pathway membership rather than ranked gene list is used for comparison

N-Acetyl Cysteine May Support Dopamine Neurons in Parkinson\u27s Disease: Preliminary Clinical and Cell Line Data.

BACKGOUND: The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson\u27s disease (PD). METHODS: The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD, using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study, patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson\u27s Disease Rating Scale (UPDRS) to measure clinical symptoms. RESULTS: The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC, consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; p CONCLUSIONS: The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients, and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT02445651