Gram‐positive bacteria produce membrane vesicles: Proteomics‐based characterization of Staphylococcus aureus‐derived membrane vesicles

Although archaea, Gram‐negative bacteria, and mammalian cells constitutively secrete membrane vesicles (MVs) as a mechanism for cell‐free intercellular communication, this cellular process has been overlooked in Gram‐positive bacteria. Here, we found for the first time that Gram‐positive bacteria naturally produce MVs into the extracellular milieu. Further characterizations showed that the density and size of Staphylococcus aureus‐derived MVs are both similar to those of Gram‐negative bacteria. With a proteomics approach, we identified with high confidence a total of 90 protein components of S. aureus‐derived MVs. In the group of identified proteins, the highly enriched extracellular proteins suggested that a specific sorting mechanism for vesicular proteins exists. We also identified proteins that facilitate the transfer of proteins to other bacteria, as well to eliminate competing organisms, antibiotic resistance, pathological functions in systemic infections, and MV biogenesis. Taken together, these observations suggest that the secretion of MVs is an evolutionally conserved, universal process that occurs from simple organisms to complex multicellular organisms. This information will help us not only to elucidate the biogenesis and functions of MVs, but also to develop therapeutic tools for vaccines, diagnosis, and antibiotics effective against pathogenic strains of Gram‐positive bacteria.     

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.     

Compressive mechanical force augments osteoclastogenesis by bone marrow macrophages through activation of c‐Fms‐mediated signaling

Little is known about the effects of mechanical forces on osteoclastogenesis by bone marrow macrophages (BMMs) in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study, we examined the effects of mechanical force on osteoclastogenesis by applying centrifugal force to BMMs using a horizontal microplate rotor. Our findings, as measured by an in vitro model system, show that tumor necrosis factor (TNF)‐α is capable of inducing osteoclast differentiation from BMMs and bone resorption in the presence of macrophage‐colony stimulating factor (M‐CSF) and is further facilitated by receptor activator of nuclear factor‐kappaB (NF‐κB) ligand (RANKL). Application of force to BMMs accelerated TNF‐α‐induced osteoclastogenesis; this was inhibited either by anti‐TNF‐α or anti‐TNF‐α receptor but not by OPG. TNF‐α also increased c‐Fms expression at both mRNA and protein levels in BMMs. An anti‐c‐Fms antibody completely inhibited osteoclast differentiation and bone resorption induced by TNF‐α but partially blocked osteoclastogenesis stimulated in combination with RANKL. These results suggest that TNF‐α (in the presence of M‐CSF) is capable of inducing osteoclastogenesis from BMMs, and that osteoclastogenesis is significantly stimulated by force application through the activation of c‐Fms‐mediated signaling. Overall, the present study reveals the facilitating effect of mechanical force on osteoclastic differentiation from BMMs without the addition of mechanosensitive cells. J. Cell. Biochem. 111: 1260–1269, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Transient Cerebral Ischemia Induces Active Astrocytosis Without Distinct Neuronal Death in the Gerbil Main Olfactory Bulb: A Long-Term Analysis

In the present study, we examined ischemia-induced neuronal and glial changes in the gerbil MOB at various time points during 60 days after 5 min of transient cerebral ischemia. The number of neuronal neuclei-immunoreactive neurons was not changed after ischemia/reperfusion (I/R). Myelin basic protein immunoreaction was well preserved after I/R. Five days after I/R, reactive form of GFAP-immunoreactive astrocytes began to increase in the external plexiform layer and granule cell layer: These reactive astrocytes peaked 10 days after I/R, thereafter, they decreased with time after I/R. Iba-1-immunoreactive microglia were ubiquitously distributed in all layers of the MOB. After I/R, significant changes in their morphology and immunoreactivity were not detected. The results of western blot analyses for GFAP, Iba-1 and MBP were similar to the immunohistochemical data. In addition, 8-hydroxy-2′-deoxyguanosine (a marker for DNA damage) immunoreactivity and SOD1, an antioxidant, protein levels were not changed in the ischemic MOB. These results indicate that neurons in the MOB are resistant to ischemic insult, showing that astrocytes are activated late in the ischemic MOB.     

Identification of Differentially Expressed Genes in Flower Buds of <Emphasis Type="Italic">Calanthe discolor</Emphasis> and <Emphasis Type="Italic">C. sieboldii</Emphasis>

The genus Calanthe includes species of terrestrial orchids that produce attractive flowers with diverse floral traits. Breeding programs have been established to improve the horticultural value of various Calanthe species, but studies to identify the genetic components contributing to the key phenotypic characteristics have not been undertaken. To understand the molecular mechanisms underlying floral development associated with floral morphology, color, and fragrance production, the flower buds of two typical Korean Calanthe species, C. discolor and C. sieboldii, were subjected to gene expression analysis by differential display RT-PCR (DDRT-PCR). A total of 66 non-redundant differentially expressed genes (DEGs) were isolated and sequenced. Of these, 26 and 40 DEGs were found to be highly expressed in C. discolor and C. sieboldii, respectively. Moreover, the expression patterns of a subset of genes presumably implicated in signal transduction, metabolic pathways, and hormonal signaling differed between the two species. The data presented here may improve our understanding of the mechanisms underlying floral development and contribute to advances in orchid biotechnology.     

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.     

Side-chain homologation of nodulisporic acid: synthesis of potent new dienyl derivatives

A series of new, diene-modified nodulisporic acid analogues (2) bearing diverse functionality at the 3"- and 4"-sites was efficiently prepared from the 3"-aldehyde 3. Biological evaluation of these synthetic nodulisporic acid analogues for systemic flea efficacy identified potent compounds and further clarified the structural requirements for ectoparasite activity.