ApoE receptor 2 controls neuronal survival in the adult brain

A central pathogenic feature of neurodegenerative diseases and neurotrauma is the death of neurons. A mechanistic understanding of the factors and conditions that induce the dysfunction and death of neurons is essential for devising effective treatment strategies against neuronal loss after trauma or during aging. Because Apolipoprotein E (ApoE) is a major risk factor for several neurodegenerative diseases, including Alzheimer's disease , a direct or indirect role of ApoE receptors in the disease process is likely. Here we have used gene targeting in mice to investigate possible roles of ApoE receptors in the regulation of neuronal survival. We demonstrate that a differentially spliced isoform of an ApoE receptor, ApoE receptor 2 (Apoer2), is essential for protection against neuronal cell loss during normal aging. Furthermore, the same splice form selectively promotes neuronal cell death after injury through mechanisms that may involve serine/threonine kinases of the Jun N-terminal kinase (JNK) family. These findings raise the possibility that ApoE and its receptors cooperatively regulate common mechanisms that are essential to neuronal survival in the adult brain.     

Norepinephrine activates store-operated Ca2+ entry coupled to large-conductance Ca2+-activated K+ channels in rat pinealocytes

Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in the pineal gland. Although a substantial amount of Ca²⁺ influx is triggered by NE, the Ca²⁺ entry pathway and its physiological relevance have not been elucidated adequately. Herein we report that the Ca²⁺ influx triggered by NE significantly regulates the protein level of serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and is responsible for maintaining the Ca²⁺ response after repetitive stimulation. Ca²⁺ entry evoked by NE was dependent on PLC activation. NE evoked a substantial amount of Ca²⁺ entry even after cells were treated with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analog of diacylglycerol. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca²⁺ entry. Moreover, NE failed to induce further Ca²⁺ entry after the development of Ca²⁺ entry induced by thapsigargin (Tg), suggesting that the pathway of Ca²⁺ entry induced by NE might be identical to that of Tg. Interestingly, Ca²⁺ entry evoked by NE or Tg induced membrane hyperpolarization that was reversed by iberiotoxin (IBTX), a specific inhibitor of large-conductance Ca²⁺-activated K⁺ (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, the activation of BK channels triggered by NE contributed to regulation of the protein level of AANAT. Collectively, these results suggest that NE triggers Ca²⁺ entry coupled to BK channels and that NE-induced Ca²⁺ entry is important in the regulation of AANAT. serotonin N-acetyltransferase; pineal gland     

Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root

Phospholipase A₂ (PLA₂), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA₂s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA₂α, a PLA₂ isoform, colocalized with the Golgi marker. Impairments of PLA₂ function by PLA₂α mutation, PLA₂-RNA interference (RNAi), or PLA₂ inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA₂ hydrolytic product) restored the PM localization of PINs in the pla₂α mutant and the ONO-RS-082–treated seedling. Suppression of PLA₂ activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair–specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA₂ inhibitor treatments, PLA₂α mutation, or PLA₂-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA₂, likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.     

Dogs cloned from fetal fibroblasts by nuclear transfer

Fetal fibroblasts have been considered as the prime candidate donor cells for the canine reproductive cloning by somatic cell nuclear transfer (SCNT) in regard to the future production of transgenic dogs, mainly due to their higher developmental competence and handling advantage in gene targeting. In this study, the cloning efficiency with canine fetal fibroblasts as donor cells was determined. A total of 50 presumptive cloned embryos were reconstructed, activated and transferred into the oviducts of naturally synchronous recipient bitches. While the fusion rate (76.9%) was similar to those of our earlier studies with adult fibroblasts as donor cells (73.9–77.1%), a high cloning efficiency (4.0%; 2 births/50 embryos transferred) was found compared to the previous success rate with adult fibroblasts (0.2–1.8%). The cloned beagles were healthy and genotypically identical to the donor fibroblast cells. This study shows that a fetal fibroblast cell would be an excellent donor for future production of transgenic dogs via gene targeting in this cell followed cloning using SCNT technology.     

Secretory phospholipase A2 promotes MMP‐9‐mediated cell death by degrading type I collagen via the ERK pathway at an early stage of chondrogenesis

Background information. sPLA₂ (secretory phospholipase A₂) has been implicated in a wide range of cellular responses, including cell proliferation and ECM (extracellular matrix) remodelling. Even though ECM remodelling is an essential step for chondrogenesis, the expression and functions of sPLA₂ during chondrogenesis have not been studied. Results. In the present study, for the first time, we detect the secretion of sPLA₂ during limb development and suggest that sPLA₂ influences the proliferation and/or survival of limb mesenchymal cells. Treatment of wing bud mesenchymal cells with exogenous sPLA₂ promoted cell death by activating MMP‐9 (matrix metalloproteinase‐9) and increasing type I collagen degradation. The additive chondro‐inhibitory actions were induced by co‐treatment of mp‐BSA (p‐aminophenyl‐mannopyranoside‐BSA), a known ligand of the mannose receptor. Chondro‐inhibitory actions by sPLA₂ were prevented by functional blocking of FcRY (chicken yolk sac IgY receptor), a mannose receptor family member that is the orthologue of the mammalian PLA₂ (phospholipase A₂) receptor and by inhibition of ERK (extracellular‐signal‐regulated kinase) activity. Conclusions. Taken together, our results suggest that elevated levels of sPLA₂ secreted by wing bud mesenchymal cells promote type I collagen degradation by MMP‐9 in a manner typical of receptor‐mediated signalling and that these events lead to cell death.     

Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge,Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting

To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.Enhanced biological phosphorus removal (EBPR) has been applied in many wastewater treatment plants to reduce the phosphorus that causes eutrophication in surface waters. EBPR employs polyphosphate-accumulating organisms (PAOs), which are enriched through alternating aerobic-anaerobic cycles (34). Since PAOs are essential for an understanding of EBPR, many candidates have been proposed as potential PAOs, such as Acinetobacter spp. (11), Tetrasphaera spp. (31), Microlunatus phosphovorus (36), Lampropedia spp. (40), and Gram-positive Actinobacteria (24). However, those organisms do not exhibit all of the characteristics of the EBPR biochemistry model. Recently developed culture-independent approaches such as PCR-clone libraries, fluorescence in situ hybridization (FISH), and microautoradiography (MAR) have highlighted an uncultured Rhodocyclus-related bacterium, “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), as one of the most important PAO candidates (2, 5, 16, 22, 23, 27, 28, 47).Numerous studies have sought to investigate uncultured “Ca. Accumulibacter” and have shown the presence of genetically and physiologically different members with a global geographic distribution (3, 9, 22, 27, 39). For example, Kong et al. (22) identified two morphologically different “Ca. Accumulibacter” cells of small cocci and large coccobacilli labeled with PAOmix (PAO462, PAO651, and PAO846) in laboratory-scale EBPR reactors. Additional results showing phenotypic and morphological diversities of “Ca. Accumulibacter” cells also existed with respect to the different roles of denitrifying PAO (DPAO) in the EBPR process (3, 9, 23). Carvalho et al. (3) detected two different morphotypes of “Ca. Accumulibacter” with different nitrate reduction capabilities. The presence of other “Ca. Accumulibacter” strains with 15% genome sequence divergence from the dominant strains in metagenomic analyses is likely to explain these morphological and phenotypic differences (12). McMahon et al. (33) suggested the use of the polyphosphate kinase (ppk) gene, which is involved in the production of polyphosphate, for a finer elucidation of “Ca. Accumulibacter” diversity. He et al. (15) grouped “Ca. Accumulibacter” strains into five distinct clades, designated clades I, IIA, IIB, IIC, and IID, using ppk gene sequence information. Flowers and colleagues (9) previously reported that “Ca. Accumulibacter” cells of clade IA had nitrate reduction activity with phosphorus uptake but that “Ca. Accumulibacter” cells of clade IIA did not.FISH-fluorescence activated cell sorting (FACS) techniques have been used for the separation of specific microbial cells from complex microbial consortia and their metabolic gene analysis (14, 46). For example, Miyauchi et al. (35) sorted PAOmix probe-labeled “Ca. Accumulibacter” cells from EBPR sludge and analyzed their nitrite reductase gene (nirS) diversity. In the current study, we found that four different “Ca. Accumulibacter” clades (Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4) were present in the EBPR sludge of laboratory-scale reactors supplied with acetate as the sole carbon source. We analyzed their morphological characteristics and ppk gene sequence information using a suite of FISH and FACS approaches and linked fine-scale phylogenetic diversities of “Ca. Accumulibacter” strains with their morphological characteristics and metabolic genes. This study will be useful for further genetic and physiological studies of different “Ca. Accumulibacter” clades.     

Relation Among Neuronal Death,Cell Proliferation and Neuronal Differentiation in the Gerbil Main Olfactory Bulb after Transient Cerebral Ischemia

Neurogenesis occurs during the embryonic stage and throughout life. Brain injuries such as ischemic insults enhance cell proliferation in some areas of the brain. We examined proliferation of newly generated cells in each layer of the gerbil main olfactory bulb (MOB) after 5 min of transient cerebral ischemia using bromodeoxyuridine (BrdU) immunohistochemistry. Ischemia-related neuronal death in the MOB was not detected using Fluoro-Jade B histofluorescence and TUNEL staining. Many BrdU-positive (⁺) cells were found in the rostral migratory stream in control and ischemic MOBs. Significant increase of BrdU⁺ cells was observed in the granule cell layer (GCL) and glomerular layer (GL) from 15 days post-ischemia, and BrdU⁺ cells were very much higher than those of the control group 30 days post-ischemia. At this time point after ischemia/reperfusion, a few BrdU⁺ cells in the GL and GCL were co-localized with calretinin⁺ cells, and many BrdU⁺ cells expressed doublecortin, a marker of immature neurons. These results indicate that cell proliferation is increased in the GCL and GL without apparent neuronal loss from 15 days after transient cerebral ischemia in gerbils.