Victorin triggers programmed cell death and the defense response via interaction with a cell surface mediator

The host-selective toxin victorin is produced by Cochliobolus victoriae, the causal agent of victoria blight of oats. Victorin has been shown to bind to the P protein of the glycine decarboxylase complex (GDC) in mitochondria, and induce defense-related responses such as phytoalexin synthesis, extracellular alkalization and programmed cell death. However, evidence demonstrating that the GDC plays a critical role in the onset of cell death is still lacking, and the role of defense-like responses in the pathogenicity has yet to be elucidated. Here, cytofluorimetric analyses, using the fluorescein (VicFluor) or bovine serum albumin-fluorescein derivative of victorin (VicBSA), demonstrated that victorin-induced cell death occurs before these conjugates traverse the plasma membrane. As with native victorin, VicBSA clearly elicits apoptosis-like cell death, production of phytoalexin, extracellular alkalization, and generation of nitric oxide and reactive oxygen intermediates. These results suggest that the initial recognition of victorin takes place on the cell surface, not in mitochondria, and leads to the activation of a battery of victorin-induced responses. Pharmacological studies showed that extracellular alkalization is the essential regulator for both victorin- and VicBSA-induced cellular responses. We propose a model where victorin may kill the host cell by activating an HR-like response, independent of the binding to the GDC, through ion fluxes across the plasma membrane.     

Protective effect of egg yolk phosvitin against ultraviolet-light-induced lipid peroxidation in the presence of iron ions

It is known that nonheme iron accumulates and free radicals are generated in skin exposed to ultraviolet (UV) light. Iron ions have a role in skin photodamage by participating in the formation of reactive oxygen species. In this study, we evaluated the effect of egg yolk phosvitin on UV-light-induced oxidative stress. Mouse dorsal skin homogenate was exposed to UVA light in the presence or absence of ferric nitrilotriacetate, (FeNTA). Lipid peroxidation was determined by measuring thiobarbituric acid-reactive substances (TBARS). The TBARS concentration increased with increasing FeNTA concentration and UV-light-exposure time. In the presence of FeNTA, phosvitin more effectively inhibited in vitro lipid peroxidation than did bovine serum albumin. According to results of electron spin resonance studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent, phosvitin suppressed the formation of hydroxyl radicals. These results suggest that UV-light-induced oxidative stress can be reduced by phosvitin.     

Reverse pharmacology of orexin: from an orphan GPCR to integrative physiology

Orexins, which were initially identified as endogenous peptide ligands for two orphan G-protein coupled receptors (GPCRs), have been shown to have an important role in the regulation of energy homeostasis. Furthermore, the discovery of orexin deficiency in narcolepsy patients indicated that orexins are highly important factors for the sleep/wakefulness regulation. The efferent and afferent systems of orexin-producing neurons suggest interactions between these cells and arousal centers in the brainstem as well as important feeding centers in the hypothalamus. Electrophysiological studies have shown that orexin neurons are regulated by humoral factors, including leptin, glucose, and ghrelin as well as monoamines and acetylcholin. Thus, orexin neurons have functional interactions with hypothalamic feeding pathways and monoaminergic/cholinergic centers to provide a link between peripheral energy balance and the CNS mechanisms that coordinate sleep/wakefulness states and motivated behavior such as food seeking.     

Shared molecular characteristics of successfully transformed mitochondrial genomes in <Emphasis Type="Italic">Chlamydomonas</Emphasis><Emphasis Type="Italic">reinhardtii</Emphasis>

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.     

记中国首次发现的"真古兽类" (eupantotherian)化石

记述了在中国首次发现的采自辽宁黑山县八道壕矿区早白垩世晚期沙海组的一件“eu- pantotherian”(“真古兽类”)下颌骨化石。标本保存了最后两个前臼齿和4个臼齿,它以抬高的下颌角突,半臼齿化的最后一枚前臼齿,臼齿上面积增大但未发育成完整盆形的跟座,尚未形成的facet-5,及加长的最后臼齿等特点有别于所有已知的“eupantotherian”和具有雏形磨楔式臼齿的Kielantherium,被命名为一新属新种,Mozomus shikamai gen.et sp.nov.(鹿间明镇古兽),并由它而创建了一新科,Mozomuridae fam.nov. “Eupantotherian”是早期哺乳动物演化中的一个重要环节,是从无跟座的对齿兽(sym- metrodont)到具有磨楔式(tribosphenic)臼齿兽类的中间类型。早期兽类进化的成功模式是发育成具有磨楔式的臼齿,即在上臼齿上发育出原尖,而下臼齿的跟座形成由3个齿尖围成的盆状。这种结构扩大了牙齿的面积,使咀嚼切割能力更趋完善,今天的有袋类和真兽类均是如此。但在哺乳动物系统发育史上,“eupantotherian”类的化石发现不多,这给探讨具有磨楔式臼齿构造的两大门类(后兽类和真兽类)的起源带来不少困难和疑惑。而传统上的真古兽类形态分异又很大,并不是一个单系类群。其中有跟座发育较好者,如peramurans有可能更接近具有磨楔式臼齿兽类的基部位置,本文记述的Mozomus shikamai也应属于这一类型。具有雏形的被认为处于基干上的磨楔式臼齿类化石,迄今只有两种,即发现在英国早白垩世地层中的滨齿兽(Aegialodon)和蒙古早白垩世晚期Hoobor层的Kielantherium,前者仅有一颗下臼齿,后者由一枚下臼齿和一具有4颗臼齿的下牙床为代表。两种化石在分类上被归入单一的滨齿兽目(Order Aegialodontia Butler,1978),视为Boreosphenidans的基干(stem)。本文记述的Mozomus,其时代与Kielantherium的大体相当,在大小、齿式及臼齿形态上与后者也多有相近之处,但前者以其臼齿的facet-5尚未出现和跟盆发育不全等特点表明它较Kielantherium更为原始,不具备磨楔式臼齿的模式,因之不能归人Aegialodontia,而只能纳入”eupantotheri- ans”。但在后一类的组合中,Mozomus以它半臼齿化的最后前臼齿和面积增大但未发育成盆形的跟座等特点,又是组合中相当进步的类型。无论如何Mozomus的发现是在为数极少的向磨楔式臼齿模式进化的中间环节上增添了一件重要的化石标本,也增加了不少新的信息。它必会引起学者对这一进化过程的更加深入的反思和新的启示。     

Molecular cloning and functional expression of hemolysin from the sea anemone Actineria villosa

The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cysteine residues and Arg-Gly-Asp (RGD) motif sequence. The mature Avt-I has a predicted molecular weight of 19.6 kDa and its theoretical isoelectric point is 9.3. The Avt-I revealed 99, 61, 57, and 57% amino acid similarity with hemolytic toxins Pstx20, EqtII, StII, and HmT from Phyllodiscus semoni, Actinia Equina, Stichodactyla helianthus, and Heteractis magnifica, respectively. The characteristic amphiphilic alpha-helix structure was found at the N-terminal region of the mature Avt-I. Recombinant Avt-I (rAvt-I) was expressed in Escherichia coli BL21 (DE3) strain as a biologically active form and purified rAvt-I caused 50% hemolytic activity against 1% sheep erythrocytes at a concentration of 6.3 ng/ml (0.32 nM). M9Y medium led to more than 2-fold increase in rAvt-I yield than cultivation in Luria-Bertani medium.     

Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells

Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes.     

A sorting nexin PpAtg24 regulates vacuolar membrane dynamics during pexophagy via binding to phosphatidylinositol-3-phosphate

Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.