A new thermolabile alkaline phospholipase D from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS628

A phospholipase D (PLD₆₂₈), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7 ∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD₆₂₈ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD₆₂₈ lacks the transphosphatidylation activity. PLD₆₂₈ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.     

Oxidative damage in Parkinson disease: Measurement using accurate biomarkers

Oxidative damage has been implicated in the pathogenesis of Parkinson disease (PD) but the literature data are confusing. Using products of lipid and DNA oxidation measured by accurate methods, we assessed the extent of oxidative damage in PD patients. The levels of plasma F₂-isoprostanes (F₂-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), cholesterol oxidation products, neuroprostanes (F₄-NPs), phospholipase A₂ (PLA₂) and platelet activating factor–acetylhydrolase (PAF-AH) activities, urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG), and serum high-sensitivity C-reactive protein were compared in 61 PD patients and 61 age-matched controls. The levels of plasma F₂-IsoPs, HETEs, 7β-and 27-hydroxycholesterol, 7-ketocholesterol, F₄-NPs, and urinary 8-OHdG were elevated, whereas the levels of plasma PLA₂ and PAF-AH activities were lower, in PD patients compared to controls (p <  0.05). The levels of plasma F₂-IsoPs, HETEs, and urinary 8-OHdG were higher in the early stages of PD (p trend <  0.05). There was a significant negative correlation between the cumulative intake of levodopa and urinary 8-OHdG (r =  ?0.305, p =  0.023) and plasma total HETEs (r =  ?0.285, p =  0.043). Oxidative damage markers are systemically elevated in PD, which may give clues about the relation of oxidative damage to the onset and progression of PD.     

Nigella sativa thymoquinone-rich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats

The antioxidant activities of the thymoquinone-rich fraction (TQRF) extracted from Nigella sativa and its bioactive compound, thymoquinone (TQ), in rats with induced hypercholesterolemia were investigated. Rats were fed a semipurified diet supplemented with 1% (w/w) cholesterol and were treated with TQRF and TQ at dosages ranging from 0.5 to 1.5 g/kg and 20 to 100 mg/kg body wt, respectively, for 8 weeks. The hydroxyl radical (OH^·)-scavenging activity of plasma samples collected from experimental rats was measured by electron spin resonance. The GenomeLab Genetic Analysis System was used to study the molecular mechanism that mediates the antioxidative properties of TQRF and TQ. Plasma total cholesterol and low-density-lipoprotein cholesterol levels were significantly decreased in the TQRF- and TQ-treated rats compared to untreated rats. Feeding rats a 1% cholesterol diet for 8 weeks resulted in a significant decrease in plasma antioxidant capacity, as measured by the capacity to scavenge hydroxyl radicals. However, rats treated with TQRF and TQ at various doses showed significant inhibitory activity toward the formation of OH^· compared to untreated rats. Upon examination of liver RNA expression levels, treatment with TQRF and TQ caused the up-regulation of the superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 2 (GPX) genes compared to untreated rats (P < 0.05). In support of this, liver antioxidant enzyme levels, including SOD1 and GPX, were also apparently increased in the TQRF- and TQ-treated rats compared to untreated rats (P < 0.05). In conclusion, TQRF and TQ effectively improved the plasma and liver antioxidant capacity and enhanced the expression of liver antioxidant genes of hypercholesterolemic rats.     

Leucine-rich repeat kinase 2 induces α-synuclein expression via the extracellular signal-regulated kinase pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.     

Dishevelled: The hub of Wnt signaling

Wnt signaling controls a variety of developmental and homeostatic events. As a key component of Wnt signaling, Dishevelled (Dvl/Dsh) protein relays Wnt signals from receptors to downstream effectors. In the canonical Wnt pathway that depends on the nuclear translocation of β-catenin, Dvl is recruited by the receptor Frizzled and prevents the constitutive destruction of cytosolic β-catenin. In the non-canonical Wnt pathways such as Wnt-Frizzled/PCP (planar cell polarity) signaling, Dvl signals via the Daam1-RhoA axis and the Rac1 axis. In addition, Dvl plays important roles in Wnt-GSK3β-microtubule signaling, Wnt-calcium signaling, Wnt-RYK signaling, Wnt-atypical PKC signaling, etc. Dvl also functions to mediate receptor endocytosis. To fulfill its multifaceted functions, it is not surprising that Dvl associates with various kinds of proteins. Its activity is also modulated dynamically by phosphorylation, ubiquitination and degradation. In this review, we summarize the current understanding of Dvl functions in Wnt signal transduction and its biological functions in mouse development, and also discuss the molecular mechanisms of its actions.     

Value of human amniotic epithelial cells in tissue engineering for cornea

Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.     

Development of a treatment solution for reductive dechlorination of hexachloro-1,3-butadiene in vadose zone soil

The biodegradation of chlorinated organics in vadose zone soils is challenging owing to the presence of oxygen, which inhibits reductive dehalogenation reactions and consequently the growth of dehalorespiring microbes. In addition, the hydraulic conductivity of vadose zone soils is typically high, hence attempts to remediate such zones with biostimulation solutions are often unsuccessful due to the short residence times for these solutions to act upon the native bacterial community. In this study we have identified sodium alginate as a hydrogel polymer that can be used to increase the residence time of a nutrient solution in an unsaturated sandy soil. Additionally we have identified neutral red as a redox active compound that can catalyse the reductive dechlorination of the chlorinated organic hexachloro-1,3-butadiene by activated sludge fed with lactate and acetate. Finally we have shown that a nutrient solution amended with neutral red and sodium alginate can lower the redox potential and reduce hexachloro-1,3-butadiene concentrations in a contaminated vadose zone soil.     

Formononetin,an isoflavone,relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 μM) and methylene blue (10 μM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS^Ser1177 protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 μM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 μM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 μM, an estrogen receptor (ERα/ERβ) antagonist) or mifepristone (10 μM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca²⁺-activated K⁺ (BK_Ca) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K⁺ (K_ATP) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK_Ca and K_ATP channels.     

Novel pyrimidines as acid pump antagonists (APAs)

A series of pyrimidine derivatives as acid pump antagonists (APAs) was synthesized and the inhibitory activities against H⁺/K⁺ ATPase isolated from hog gastric mucosa were determined. After elaborating on substituents at C2 and C4 position of the pyrimidine scaffold, we have observed that the compound 7h is a potent APA with H⁺/K⁺ ATPase, IC₅₀ = 52 nM.