Carotenoids are produced by a variety of organisms, but the mechanisms that regulate gene expression leading to carotenoid biosynthesis have been characterized for only a few organisms. In this study, we found that Streptomyces coelicolor A3(2), a gram-positive filamentous bacterium, produces carotenoids under blue light induction. The carotenoid fraction isolated from the cell extract contained multiple compounds, including isorenieratene and ␤-carotene. The carotenoid biosynthesis gene cluster of S. coelicolor consists of two convergent operons, crtEIBV and crtYTU, as previously shown for Streptomyces griseus. The crtEIBV null mutant completely lost its ability to produce carotenoids. The crt gene cluster is flanked by a regulatory region that consists of two divergent operons, litRQ and litSAB. The lit (light-induced transcription) genes encode a MerR-type transcriptional regulator (LitR), a possible oxidoreductase (LitQ), an extracytoplasmic function sigma factor ( LitS ), a putative lipoprotein (LitA), and a putative anti-sigma factor (LitB). S1 protection assay revealed that the promoters preceding crtE (PcrtE), crtY (PcrtY), litR (PlitR), and litS (PlitS) are activated upon illumination. A litS mutant lost both the ability to produce carotenoids and the activities of PcrtE, PcrtY, and PlitS, which suggested that LitS directs light-induced transcription from these promoters. An RNA polymerase holocomplex containing purified LitS recombinant protein generated specific PcrtE and PcrtY transcripts in an in vitro runoff transcriptional assay. A litR mutant that had an insertion of the kanamycin resistance gene was defective both in the ability to produce carotenoids and in all of the light-dependent promoter activities. Overexpression of litS resulted in constitutive carotenoid production in both the wild type and the litR mutant. These results indicate that LitS acts as a light-induced sigma factor that directs transcription of the crt biosynthesis gene cluster, whose activity is controlled by an unknown LitR function. This is the first report to describe light-inducible gene expression in Streptomyces.

Parkinson disease (PD) is the second most common neurodegenerative disease. The muscle rigidity, tremor, and bradykinesia that are characteristic of PD patients, are caused by dopaminergic neuron death in the substantia nigra. One of the well-known pathological hallmarks inside the cells is the presence of inclusion bodies called Lewy bodies (LB) that include aggregates of a-synuclein. Since a-synuclein and leucine-rich repeat kinase 2 (LRRK2) cause familial forms of PD that resembles sporadic PD pathologically, these genetic mutations provide important molecular tools to investigate PD pathogenesis. 1) Although the etiology of PD remains unclear, the production of aggregated a-synuclein is a key step in PD pathogenesis. 2)a-Synuclein plays an important role in PD pathology and neuronal cell death. [3][4][5] Point mutations (A30P, E46K, and A53T) and multiplication of the gene a-synuclein are linked to the early-onset of PD pathology. The increased severity of PD and earlier age of onset have been reported to correlate with increased a-synuclein dosage.6) The physiological function of a-synuclein found in pre-synapus remains undefined. Several studies have indicated that a-synuclein regulates intracellular transport of synaptic vesicles underlying neurotransmitter release.7-14) a-Synuclein may also be involved in mitochondrial complex I function, 15,16) and has been reported to impair macroautophagy. 17)Until recently, a-synuclein was considered to exert pathogenic effects inside the cells. However, a-synuclein can be detected in human cerebrospinal fluid (CSF) and plasma.18)The accumulation of aggregated a-synuclein spreads from lower brainstem into the limbic system and neocortex, suggesting a mechanism underlying pathological propagation of PD such as Prion diseases.19) Some groups have reported that aggregated a-synuclein can be released into extracellular media by exocytosis through exosomes and propagated by direct neuron-to-neuron transmission. [20][21][22] LRRK2 is a large 2527 amino acid protein consisting of several functional domains including a Ras-like small GTPase domain (ROC), a carboxy-terminal of Roc (COR) domain, and a kinase domain. The various mutations in LRRK2 are involved in PD, such as R1441C, R1441G in the Roc domain, Y1699C in the COR domain, and G2019S, I2020T in the kinase domain. Among them, G2019S mutation clearly increases kinase activity, which is required for PD pathology.23,24) G2019S mutation has been shown to increase kinase activity by 2 to 3 fold. However, normal function and kinase substrates of LRRK2 remain unclear. Although mutant LRRK2 was toxic when overexpressed in cultured cells 25,26) and Drosophila, 27,28) loss of neurons was not observed in transgenic mice overexpressing R1441G and R1441C mutants. 29,30) Loss of LRRK2 did not cause neurodegeneration and neuropathological changes. LRRK2 mutations cause clinically typical PD features, ranging from nigral degeneration without LB to nigral degeneration with widespread LB or neurofibrillary tangles. 31)The neuronal cell...

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a lung disease with high mortality. In addition, osteonecrosis and bone abnormalities with reduced bone density have been observed in patients following recovery from SARS, which were partly but not entirely explained by the short-term use of steroids. Here, we demonstrate that human monocytes, potential precursors of osteoclasts, partly express angiotensin converting enzyme 2 (ACE2), a cellular receptor of SARS-CoV, and that expression of an accessory protein of SARS-CoV, 3a/X1, in murine macrophage cell line RAW264.7 cells, enhanced NF-kappaB activity and differentiation into osteoclast-like cells in the presence of receptor activator of NF-kappaB ligand (RANKL). Furthermore, human epithelial A549 cells expressed ACE2, and expression of 3a/X1 in these cells up-regulated TNF-alpha, which is known to accelerate osteoclastogenesis. 3a/X1 also enhanced RANKL expression in mouse stromal ST2 cells. These findings indicate that SARS-CoV 3a/X1 might promote osteoclastogenesis by direct and indirect mechanisms.

Poly(ADP-ribose)polymerase-1 (PARP-1) is thought to be required for apoptosis-inducing factor (AIF) release from mitochondria in caspase-independent apoptosis. The mechanism by which AIF is released through PARP-1 remains unclear. Here, we provide evidence that PARP-1-independent AIF release and cell death are induced by a trienoic fatty acid, ␣-eleostearic acid (␣-ESA). ␣-ESA induced the caspase-independent and AIF-initiated apoptotic death of neuronal cell lines, independently of PARP-1 activation. The cell death was inhibited by the MEK inhibitor U0126 and by knockdown of MEK using small interfering RNA. However, inhibitors for JNK, p38 inhibitors, calpain, phospholipase A 2 , and phosphatidylinositol 3-kinase, did not block cell death. AIF was translocated to the nucleus after the induction of apoptosis by ␣-ESA in differentiated PC12 cells without activating caspase-3 and PARP-1. The ␣-ESA-mediated cell death was not inhibited by PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinoline and by knockdown of PARP-1 using small interfering RNA. Unlike N-methyl-N-nitro-N-nitrosoguanidine treatment, histonephosphorylated histone 2AX was not phosphorylated by ␣-ESA, which suggests no DNA damage. Overexpression of Bcl-2 did not inhibit the cell death. ␣-ESA caused a small quantity of superoxide production in the mitochondria, resulting in the reduction of mitochondrial membrane potential, both of which were blocked by a trace amount of ␣-tocopherol localized in the mitochondria. Our results demonstrate that ␣-ESA induces PARP-1-independent AIF release and cell death without activating Bax, cytochrome c, and caspase-3. MEK is also a key molecule, although the link between ERK, AIF release, and cell death remains unknown. Finding molecules that regulate AIF release may be an important therapeutic target for the treatment of neuronal injury.Apoptosis is a mode of programmed cell death that is used by multicellular organisms to remove surplus and unwanted cells in the immune and nervous systems (1-5). Apoptosis is characterized by cell detachment, cell shrinkage, chromatin condensation, DNA degradation, and plasma membrane blebbing (5-7). The surplus cells are removed by caspases, which are key effector molecules of apoptotic cell death. Apoptosis is activated through two main pathways as follows: the extrinsic pathway, which originates from the activation of cell-surface death receptors, such as Fas and tumor necrosis factor-receptor 1, and results in the activation of caspase-8; and the intrinsic pathway, which originates from the mitochondrial release of cytochrome c and results in the activation of caspase-9 through the Cyt-c 2 /apoptotic protease-activating factor-1/procaspase-9 heptamer (5, 8, 9). Most apoptotic stimuli use a mitochondriondependent process such as membrane potential shutdown and outer membrane permeabilization controlled by Bax and Bak, which are pro-apoptotic members of the Bcl-2 family (6 -9). This results in the release of the pro-apoptotic protein Cyt-c, which triggers caspa...

RIP1 is a serine/threonine kinase, which is involved in apoptosis and necroptosis. In apoptosis, caspase-8 and FADD have an important role. On the other hand, RIP3 is a key molecule in necroptosis. Recently, we reported that eleostearic acid (ESA) elicits caspase-3- and PARP-1-independent cell death, although ESA-treated cells mediate typical apoptotic morphology such as chromatin condensation, plasma membrane blebbing and apoptotic body formation. The activation of caspases, Bax and PARP-1, the cleavage of AIF and the phosphorylation of histone H2AX, all of which are characteristics of typical apoptosis, do not occur in ESA-treated cells. However, the underlying mechanism remains unclear. To clarify the signaling pathways in ESA-mediated apoptosis, we investigated the functions of RIP1, MEK, ERK, as well as AIF. Using an extensive study based on molecular biology, we identified the alternative role of RIP1 in ESA-mediated apoptosis. ESA mediates RIP1-dependent apoptosis in a kinase independent manner. ESA activates serine/threonine phosphatases such as calcineurin, which induces RIP1 dephosphorylation, thereby ERK pathway is activated. Consequently, localization of AIF and ERK in the nucleus, ROS generation and ATP reduction in mitochondria are induced to disrupt mitochondrial cristae, which leads to cell death. Necrostatin (Nec)-1 blocked MEK/ERK phosphorylation and ESA-mediated apoptosis. Nec-1 inactive form (Nec1i) also impaired ESA-mediated apoptosis. Nec1 blocked the interaction of MEK with ERK upon ESA stimulation. Together, these findings provide a new finding that ERK and kinase-independent RIP1 proteins are implicated in atypical ESA-mediated apoptosis.