Molecular domain organization of BldD, an essential transcriptional regulator for developmental process of Streptomyces coelicolor A3(2)

A homodimeric protein, BldD is a key regulator for developmental process of Streptomyces coelicolor and the bldD mutant exhibits severely pleiotropic defects in the antibiotic production and morphological differentiation of the bacterium. In the present work, we approached domain organization of BldD, to structurally and functionally characterize the protein as a DNA-binding protein. We first observed a proteolytic cleavage of BldD by the cytoplasmic extracts of S. coelicolor, which was highly dependent on the developmental stage of the bacterium. The resulting fragment of BldD was identified by mass spectrometry as the N-terminal domain resistant to the proteolysis. Recombinant proteins corresponding to the intact BldD, the N-terminal domain (residues 1-79) and the rest part (C-terminal domain; residues 80-167) were used for comparative analyses by several spectroscopic, thermodynamic, and biochemical experiments, respectively. The results of circular dichroism and nuclear magnetic resonance spectroscopies certified each of the two determined domains could be regarded as an individual folding unit possessing an independent thermodynamic cooperativity. Structural interaction between the two domains was little observed in the DNA-free and DNA-bound states. Strikingly, it was revealed by gel permeation chromatography, chemical crosslink, gel mobility shift, and NMR-monitored DNA-binding experiments, that only the N-terminal domain is responsible for the dimerization as well as DNA-binding of BldD. Detailed inspection of the present results suggests that BldD function in a unique and complicated mode to totally regulate the diverse developmental stages of S. coelicolor.     

PKC delta phosphorylates p52ShcA at Ser29 to regulate ERK activation in response to H2O2

Both PKC delta and ShcA have been implicated in cell response to oxidative stress [Y. Hu, X. Wang, L. Zeng, D.Y. Cai, K. Sabapathy, S.P. Goff, E.J. Firpo, B. Li, Mol Biol Cell., 16 (2005) 3705-3718, B. Li, X. Wang, N. Rasheed, Y. Hu, S. Boast, T. Ishii, K. Nakayama, K.I. Nakayama, S.P., Goff, Genes Dev, 18 (2004) 1824-1837, E. Migliaccio, M. Giorgio, S. Mele, G. Pelicci, P. Reboldi, P.P. Pandolfi, L. Lanfrancone, P.G. Pelicci, Nature, 402 (1999) 309-313], yet their relationship in the response has not been studied. Here we report that PKC delta interacts with ShcA and this interaction is promoted by H(2)O(2). PKC delta and ShcA are also colocalized in the cytoplasm and displayed co-translocation in response to H(2)O(2). Activated PKC delta was able to phosphorylate ShcA at Ser29, as determined by mass spectrometry. These results suggest that ShcA, p66 and p52, are substrates that interact with PKC delta. This phosphorylation is critical in H(2)O(2) induced ERK activation as reconstitution with ShcA Ser29A failed to rescue ERK activation of ShcA-/- MEFs, while ShcA could. In line with this conclusion, inhibition of PKC delta with inhibitors is able to diminish H(2)O(2) induced ERK activation in MEFs. These results suggest that the interaction between PKC delta and ShcA and the phosphorylation of ShcA at Ser29 play important roles in ERK activation in cell response to H(2)O(2).     

Determination of bevantolol enantiomers in human plasma by coupled achiral-chiral high performance-liquid chromatography

A coupled achiral-chiral high performance liquid chromatographic method was developed and fully validated for the determination of bevantolol enantiomers, (-)-(S)-bevantolol and (+)-(R)-bevantolol, in human plasma. Plasma samples were prepared by solid phase extraction with Sep-Pak Plus C18 cartridges followed by HPLC. Bevantolol enantiomers and (+)-(R)-Propranolol as internal standard (IS) were preseparated from interfering components in plasma on a Phenomenex silica column and bevantolol enantiomers and IS were resolved and determined on a Chiralcel OJ-H chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The Precolumn was used to concentrate bevantolol in the eluent from the achiral column before back flushing onto chiral phase. A detailed validation of the method was performed accordingly to FDA guidelines. For each enantiomer the assay was linear between 20 and 1600 ng/ml. The quantification limits of both bevantolol enantiomers were 20 ng/ml. The intraday variation was between 1.07 and 12.64% in relation to the measured concentration and the interday variation was 0.91 and 11.79%. The method has been applied to the determination of (-)-(S)- and (+)-(R)-bevantolol in plasma from healthy volunteers dosed with racemic bevantolol hydrochloride.     

The suppressive effect of beta-glucan on the production of tumor necrosis factor-alpha in BV2 microglial cells

Beta-glucan was recently shown to have the ability to enhance and stimulate the immune system in humans, but little is known about its the anti-inflammatory effects. We investigated the effect of beta-glucan on the production of tumor necrosis factor-alpha (TNF-alpha), a major pro-inflammatory mediator, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. beta-Glucan decreased the production and expression of TNF-alpha. In addition, it blocked LPS-stimulated activation of nuclear factor kappa B (NF-kappaB). Hence beta-glucan might suppress LPS-stimulated TNF-alpha production by inhibiting NF-kappaB in BV2 microglial cells.     

Ameliorative anti-diabetic activity of dangnyosoko, a Chinese herbal medicine, in diabetic rats

The preventive anti-diabetic effect of dangnyosoko (DNSK), a Chinese herbal medicine, was evaluated in STZ-induced diabetic rats. DNSK was orally administered once a day from 3 d after STZ-induction at 100, 200, and 500 mg/kg for 4 weeks, and the results were compared to those for glibenclamide. Dramatic decreases in body weight and plasma insulin levels and increases in blood and urine glucose levels were detected in STZ-induced diabetic animals with disruption and disappearance of pancreatic islets and increases in glucagon- and decreases in insulin-producing cells. However, these diabetic changes were significantly and dose-dependently inhibited by treatment with DNSK, and DNSK at 100 mg/kg showed more favorable effects than glibenclamide at 5 mg/kg. Based on these results, it is thought that DNSK has favorable effects in ameliorating changes in blood and urine glucose levels and body weight, and that histopathological changes in the pancreas in STZ induce diabetes.     

The apoptotic effect of intercalating agents on HPV-negative cervical cancer C-33A cells

Summary. Cervical cancer is one of the leading causes of female cancer death worldwide with about 500,000 deaths per year. Both mitomycin C and cisplatin are alkylating agents, which bind and intercalate DNA, and thus used as anti-cancer drugs. In these studies, we focused on investigating the apoptotic effects of intercalating agents on HPV-negative cervical cancer C-33A cells. Accordingly, C-33A cells were treated with carboplatin, mitomycin C or cisplatin. Cell cycle analysis revealed that treatment with mitomycin C and cisplatin but not with carboplatin resulted in apoptosis. Both mitomycin C and cisplatin induced apoptosis in C-33A cells via caspase-8 and -3 processing in a Fas/FasL-dependent manner and also suppressed IL-18 expression, while they down-regulated IκB expression and up-regulated p65 expression. These results suggest that both mitomycin C and cisplatin induce apoptosis, not only via the caspase-8 and -3 dependent Fas/FasL pathway, but also via the regulation of NF-κB activity and IL-18 expression in HPV-negative cervical cancer C-33A cells.     

Caspase-11 regulates cell migration by promoting Aip1-Cofilin-mediated actin depolymerization

Coordinated regulation of cell migration, cytokine maturation and apoptosis is critical in inflammatory responses. Caspases, a family of cysteine proteases, are known to regulate cytokine maturation and apoptosis. Here, we show that caspase-11, a mammalian pro-inflammatory caspase, regulates cell migration during inflammation. Caspase-11-deficient lymphocytes exhibit a cell-autonomous migration defect in vitro and in vivo. We demonstrate that caspase-11 interacts physically and functionally with actin interacting protein 1 (Aip1), an activator of cofilin-mediated actin depolymerization. The caspase-recruitment domain (CARD) of caspase-11 interacts with the carboxy-terminal WD40 propeller domain of Aip1 to promote cofilin-mediated actin depolymerization. Cells with Aip1 or caspase-11 deficiency exhibit defects in actin dynamics. Using in vitro actin depolymerization assays, we found that caspase-11 and Aip1 work cooperatively to promote cofilin-mediated actin depolymerization. These data demonstrate a novel cell autonomous caspase-mediated mechanism that regulates actin dynamics and mammalian cell migration distinct from the receptor mediated Rho-Rac-Cdc42 pathway.     

The variety of cytosolic calcium responses and possible roles of PLC and PKC

A model of ligand-induced intracellular calcium (Ca2+) responses incorporating phospholipase C (PLC) and protein kinase C (PKC) is developed for the purpose of understanding the mechanisms underlying the observed temporal patterns of intracellular calcium (Ca(i)2+) under sustained agonist stimulation. Some studies have suggested that inhibition of ligand receptors and PLC by PKC could generate sinusoidal Ca2+ oscillations, while PKC-independent Ca2+-induced Ca2+ release (CICR) via IP(3)-gated Ca2+ channels on the endoplasmic reticulum (ER) is believed to be responsible for baseline spiking. However, some evidence also indicates that baseline spiking can be observed under high-PKC activity, or under low-PKC activity with low agonist stimulus, as well. Insight into the basis of these observations regarding the role of PKC in Ca(i)2+ response patterns can be gained by developing and analyzing a mathematical model of Ca(i)2+ responses. We do this herein and find that (1) interaction of CICR and the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump is enough to generate both types of Ca(i)2+ oscillations, (2) there exist four possible Ca(i)2+ response patterns under sustained agonist stimulus: a sub-threshold response (SR), baseline spiking, sinusoidal oscillations (SO) and transient with plateau, and (3) the IP(3) concentration, which is controlled by the strength of the interaction between PKC and PLC, can be used to predict the Ca(i)2+ response patterns. From this analysis we conclude that the different patterns of Ca(i)2+ oscillations can be understood as a generic consequence of the interactions between CICR via the IP(3)-gated Ca(2+) channels in response to changes in the level of IP(3), and re-uptake into the ER/SR via the SERCA pump. PKC, in conjunction with PLC, can act as a switch between different Ca(i)2+ response patterns by modulating the cytosolic IP(3) level, which determines the Ca(i)2+ patterns.