S-nitrosylation of NADPH oxidase regulates cell death in plant immunity

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.     

Glutamate induces de novo growth of functional spines in developing cortex

Mature cortical pyramidal neurons receive excitatory inputs onto small protrusions emanating from their dendrites called spines. Spines undergo activity-dependent remodelling, stabilization and pruning during development, and similar structural changes can be triggered by learning and changes in sensory experiences. However, the biochemical triggers and mechanisms of de novo spine formation in the developing brain and the functional significance of new spines to neuronal connectivity are largely unknown. Here we develop an approach to induce and monitor de novo spine formation in real time using combined two-photon laser-scanning microscopy and two-photon laser uncaging of glutamate. Our data demonstrate that, in mouse cortical layer 2/3 pyramidal neurons, glutamate is sufficient to trigger de novo spine growth from the dendrite shaft in a location-specific manner. We find that glutamate-induced spinogenesis requires opening of NMDARs (N-methyl-D-aspartate-type glutamate receptors) and activation of protein kinase A (PKA) but is independent of calcium-calmodulin-dependent kinase II (CaMKII) and tyrosine kinase receptor B (TrkB) receptors. Furthermore, newly formed spines express glutamate receptors and are rapidly functional such that they transduce presynaptic activity into postsynaptic signals. Together, our data demonstrate that early neural connectivity is shaped by activity in a spatially precise manner and that nascent dendrite spines are rapidly functionally incorporated into cortical circuits.     

PPAR γ partial agonist, KR-62776, inhibits adipocyte differentiation via activation of ERK

Indenone KR-62776 acts as an agonist of PPARγ without inducing obesity in animal models and cells. X-ray crystallography reveals that the indenone occupies the binding pocket in a different manner than rosiglitazone. 2-Dimensional gel-electrophoresis showed that the expression of 42 proteins was altered more than 2.0-fold between KR-62776- or rosiglitazone-treated adipocyte cells and control cells. Rosiglitazone down-regulated the expression of ERK1/2 and suppressed the phosphorylation of ERK1/2 in these cells. However, the expression of ERK1/2 was up-regulated in KR-62776-treated cells. Phosphorylated ERK1/2, activated by indenone, affects the localization of PPARγ, suggesting a mechanism for indenone-inhibition of adipogenesis in 3T3-L1 preadipocyte cells. The preadipocyte cells are treated with ERK1/2 inhibitor PD98059, a large amount of the cells are converted to adipocyte cells. These results support the conclusion that the localization of PPARγ is one of the key factors explaining the biological responses of the ligands. Received 04 March 2009; received after revision 13 March 2009; accepted 17 March 2009     

Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-₆ integrin and anti-CD71 antibodies. RA cells express high levels of ₆ integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed ₆ integrin and ₁ integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.Received 2 July 2004; received after revision 20 August 2004; accepted 10 September 2004     

Ubiquitin-proteasome pathway as a primary defender against TRAIL-mediated cell death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death as well as expression of proinflammatory genes such as CXCL8 in malignant human astrocytoma cells. However, the molecular mechanisms that determine the fate of cells are not yet understood. The ubiquitin (Ub)-proteasome pathway regulates a wide range of cellular functions through degradation of various regulatory proteins; given this, we hypothesized that this pathway may play a central role in TRAIL-mediated signaling. We demonstrate here that inhibition of the Ub-proteasome pathway enhanced TRAIL-mediated cell death of human astrocytoma CRT-MG cells within hours by blocking degradation of active caspase-8 and -3. Proteasome inhibitors suppressed TRAIL-mediated activation of NF-B; however, inhibition of the NF-B pathway alone was not sufficient to enhance TRAIL-mediated cell death. Collectively, these results suggest that the Ub-proteasome pathway may play an important role as an antiapoptotic surveillance system by eliminating activated caspases as well as mediating NF-B-dependent signals.Received 30 December 2003; received after revision 9 February 2004; accepted 13 February 2004     

Long-term sensory deprivation prevents dendritic spine loss in primary somatosensory cortex

A substantial decrease in the number of synapses occurs in the mammalian brain from the late postnatal period until the end of life. Although experience plays an important role in modifying synaptic connectivity, its effect on this nearly lifelong synapse loss remains unknown. Here we used transcranial two-photon microscopy to visualize postsynaptic dendritic spines in layer I of the barrel cortex in transgenic mice expressing yellow fluorescent protein. We show that in young adolescent mice, long-term sensory deprivation through whisker trimming prevents net spine loss by preferentially reducing the rate of ongoing spine elimination, not by increasing the rate of spine formation. This effect of deprivation diminishes as animals mature but still persists in adulthood. Restoring sensory experience after adolescent deprivation accelerates spine elimination. Similar to sensory manipulation, the rate of spine elimination decreases after chronic blockade of NMDA (N-methyl-D-aspartate) receptors with the antagonist MK801, and accelerates after drug withdrawal. These studies of spine dynamics in the primary somatosensory cortex suggest that experience plays an important role in the net loss of synapses over most of an animal's lifespan, particularly during adolescence.     

利用杀菌剂防治松萎蔫病

松萎蔫病是一种非常复杂的病害。据报道,松材线虫(Bursaphelenchus xylophilus)及其携带的细菌诱导了该病,但是对于细菌的的作用尚存争议。笔者筛选出杀细菌剂进行了温室和林间防治松萎蔫病的实验。利用每株3年生赤松(Pinus densiflora)注射3 mg的恶喹酸,松萎蔫病防效达到71 %。利用恶喹酸或阿维菌素及两者混合物对大约20年生赤松的试验表明,3种处理均显示了很高的防效。以上结果证明伴生细菌是诱导松萎蔫病的必要因素,对松萎蔫病可利用杀细菌剂或杀细菌剂与杀线剂混合物进行防控。     

Sojucktang induces apoptosis via loss of mitochondrial membrane potential and caspase-3 activation in KLE human endometrial cancer cells

Sojucktang （SJT） has long been used for the treatment of endometrial diseases in Korea. However, the mechanisms responsible for the SJT-induced apoptosis in endometrial cancer cells remain unclear. In the present study, SJT was demonstrated to show cytotoxic effect and induce apoptotic cell death via mitochondrial regulation in KLE endometrial cancer cells. Linderae Radix, Glycyrrhizae Radix, Zedoariae Rhizoma, Trogopterorum Faeces and Agelicae Gigantis Radix were found to be the potent constituent herbs of SJT to significantly decrease the viability of KLE cells by a tetra zolium salt （XTT） assay. Apoptotic bodies were observed in SJT-treated KLE cells by 4′-6-diamidino-2-phenylindole （DAPI） and TdT-mediated-dUTP nick-end labeling （TUNEL） assay. SJT also increased sub-G1 DNA contents of the cell cycle undergoing apoptosis in a dose-dependent manner. Furthermore, it was observed that SJT activated caspase-3 and cleaved poly （ADP-ribose） polymerase （PARP）, and decreased mitochondrial membrane potential in a dose-dependent manner. Taken together, this study shows that SJT exerts anti-tumor activity against KLE endometrial cancer cells via mitochondrial dependent apoptosis induction.