Local Machinery Research Articles

Interaction of 42Sp50 with the vegetal RNA localization machinery in Xenopus laevis oocytes

Localization of a specific subset of maternal mRNAs to the vegetal cortex of Xenopus oocytes is important for the regulation of germ layer formation and germ cell development. It is driven by vegetal localization complexes that are formed with the corresponding signal sequences in the untranslated regions of the mRNAs and with a number of different so-called localization proteins. In the context of the present study, we incorporated tagged variants of the known localization protein Vg1RBP into vegetal localization complexes by means of oocyte microinjection. Immunoprecipitation of the corresponding RNPs allowed for the identification of novel Vg1RBP-associated proteins, such as the embryonic poly(A) binding protein, the Y-box RNA-packaging protein 2B and the oocyte-specific version of the elongation factor 1α (42Sp50). Incorporation of 42Sp50 into localization RNPs could be confirmed by co-immunoprecipitation of Vg1RBP and Staufen1 with myc-tagged 42Sp50. Furthermore, myc-42Sp50 was found to co-sediment with the same two proteins in large, RNAse-sensitive complexes, as well as to associate specifically with several vegetally localizing mRNAs but not with nonlocalized control RNAs. Finally, oocyte microinjection experiments reveal that 42Sp50 is a protein that shuttles between the nucleus and cytoplasm. Taken together, these observations provide evidence for a novel function of 42Sp50 in the context of vegetal mRNA transport in Xenopus oocytes.

The Fate of Synaptic Input to NG2 Glial Cells: Neurons Specifically Downregulate Transmitter Release onto Differentiating Oligodendroglial Cells

NG2-expressing oligodendrocyte precursor cells (OPCs) are ubiquitous and generate oligodendrocytes throughout the young and adult brain. Previous work has shown that virtually every NG2 cell receives synaptic input from many axons, but the meaning of this signaling is not understood. In particular, it is unclear whether neurons specifically synapse onto OPCs or whether OPCs merely trace adjacent neurotransmitter release sites and are not recognized by the presynaptic neuron. Here, we show with whole-cell recordings from distinct developmental stages of oligodendroglial cells in brain slices that synaptic input essentially disappears as soon as OPCs differentiate into premyelinating oligodendrocytes (NG2(-), DM20/PLP(+), O1(+)). Uncaging experiments and tracer loading revealed that premyelinating oligodendrocytes still express a substantial number of AMPA/kainate receptors and many processes, but spontaneous and stimulated synaptic currents are mostly absent. Nevertheless, in a minority of premyelinating cells, electrical stimulation evoked small synaptic currents with an unusual behavior: their amplitude compared well with the quantal amplitude in OPCs but they occurred asynchronously and with the remarkable latency of 40-100 ms, indicating that the presynaptic release machinery has become ineffective. Mature myelinating oligodendrocytes completely lack AMPA/kainate receptors and respond to uncaging and synaptic stimulation with glutamate transporter currents. Our data show that neurons selectively synapse onto only one of several coexisting developmental stages of glial cells and thereby indicate that neurons indeed specifically signal to OPCs and are able to modulate transmitter output by regulating the local release machinery in a manner specific to the developmental stage of the postsynaptic glial cell.

Securitisation in South Africa; the Case of the Missing Conduits

Marketable securities are the end result of the securitisation process. An often neglected implication is that this resulting security can be commercial paper, not only long-term bonds. This paper investigates the asset backed commercial paper market in South Africa and shows how it is an important part of the local securitisation machinery. In addition the founding documents of the conduits are studied to determine whether the conduits are likely to suffer from a lack of liquidity support during a crisis. South African conduits are likely to have adequately liquidity support during a crisis as legally banks must support them and the size of the support needed is unlikely to be a problem for South African banks.

Branching out: mechanisms of dendritic arborization

Type-specific dendrite morphology is a hallmark of the neuron and has important functional implications in determining what signals a neuron receives and how these signals are integrated. During the past two decades, studies on dendritic arborization neurons in Drosophila melanogaster have started to identify mechanisms of dendrite morphogenesis that may have broad applicability to vertebrate species. Transcription factors, receptor-ligand interactions, various signalling pathways, local translational machinery, cytoskeletal elements, Golgi outposts and endosomes have been identified as contributors to the organization of dendrites of individual neurons and the placement of these dendrites in the neuronal circuitry. Further insight into these mechanisms will improve our understanding of how the nervous system functions and might help to identify the underlying causes of some neurological and neurodevelopmental disorders.

Challenges in land use planning around Australian airports

The privatization of major Australian airports in the late 1990s unleashed an unprecedented development wave as corporate lessees implemented ambitious business plans. While planning and environmental regulations governing on-airport development were significantly enhanced, there has been national disquiet about a governance regime that remains under the auspices of the federal government and is not effectively integrated into state and local decision-making machinery. Tensions in major airport regions have been exacerbated by the building of highly conspicuous non-aeronautical developments approved with no determining input by local decision-makers as well as the growing pressures on off-airport locations for aviation-related development. This paper canvasses this context and overviews the evolving structure of planning controls for Australia’s privatized federal airports. A range of issues surfacing through the National Aviation Policy Review process in 2008–2009 is described.

TonB-dependent transporters and their occurrence in cyanobacteria

BackgroundDifferent iron transport systems evolved in Gram-negative bacteria during evolution. Most of the transport systems depend on outer membrane localized TonB-dependent transporters (TBDTs), a periplasma-facing TonB protein and a plasma membrane localized machinery (ExbBD). So far, iron chelators (siderophores), oligosaccharides and polypeptides have been identified as substrates of TBDTs. For iron transport, three uptake systems are defined: the lactoferrin/transferrin binding proteins, the porphyrin-dependent transporters and the siderophore-dependent transporters. However, for cyanobacteria almost nothing is known about possible TonB-dependent uptake systems for iron or other substrates.ResultsWe have screened all publicly available eubacterial genomes for sequences representing (putative) TBDTs. Based on sequence similarity, we identified 195 clusters, where elements of one cluster may possibly recognize similar substrates. For Anabaena sp. PCC 7120 we identified 22 genes as putative TBDTs covering almost all known TBDT subclasses. This is a high number of TBDTs compared to other cyanobacteria. The expression of the 22 putative TBDTs individually depends on the presence of iron, copper or nitrogen.ConclusionWe exemplified on TBDTs the power of CLANS-based classification, which demonstrates its importance for future application in systems biology. In addition, the tentative substrate assignment based on characterized proteins will stimulate the research of TBDTs in different species. For cyanobacteria, the atypical dependence of TBDT gene expression on different nutrition points to a yet unknown regulatory mechanism. In addition, we were able to clarify a hypothesis of the absence of TonB in cyanobacteria by the identification of according sequences.

Participation of Xenopus Elr-type Proteins in Vegetal mRNA Localization during Oogenesis

Directional transport of specific mRNAs is of primary biological relevance. In Xenopus oocytes, mRNA localization to the vegetal pole is important for germ layer formation and germ cell development. Using a biochemical approach, we identified Xenopus Elr-type proteins, homologs of the Hu/ELAV proteins, as novel components of the vegetal mRNA localization machinery. They bind specifically to the localization elements of several different vegetally localizing Xenopus mRNAs, and they are part of one RNP together with other localization proteins, such as Vg1RBP and XStaufen 1. Blocking Elr-type protein binding by either localization element mutagenesis or antisense morpholino oligonucleotide-mediated masking of their target RNA structures, as well as overexpression of wild type and mutant ElrB proteins, interferes with vegetal localization in Xenopus oocytes.

Being a mayor: the view from four Colombian cities

Local governments are particularly relevant to people's daily lives as they manage the infrastructure and services that directly influence quality of life. Elected mayors tend to be more responsive to the needs of the poor majority than centrally appointed mayors or mayors who are elected by fellow councillors. Colombia's municipal mayors have been elected since 1988 and local governments have been given increased responsibilities and powers to raise local revenues. In this paper, four municipal mayors at the end of their four-year term reflect, in their own words, on the role they have played in shaping their municipalities. They recount their personal engagement with local politics, share their views about their society and problems such as poverty, inequality and violence, describe the business of managing a local government and exercising leadership sometimes in difficult circumstances, reflect on the qualities of a mayor, and explain how they sought continuity for their government programmes. They see themselves as outsiders struggling against local political machineries. With one exception, they do not seem to see the poor as their natural interlocutors on whom they can rely for unswerving political support and to whom most of their energy should be directed.

Lysosomal sorting of ABC transporter TAP‐like (ABCB9)

TAP‐like (TAPL, ABCB9) is an ATP‐binding cassette (ABC) transporter with similarity to TAP, a transporter associated with antigen processing. Both are half‐type transporters of ABCB subfamily and they localize distinctly in lysosome and endoplasmic reticulum, respectively. Here, we identified a region necessary for lysosomal targeting of TAPL.PROCEDURESVarious truncated forms of TAPL tagged with GFP were stably expressed in CHO‐K1 cells. Subcellular distributions of the tagged proteins were analyzed by confocal microscopy.RESULTSTAPL has ten transmembrane helices followed by conserved ABC region. Amino‐terminal four helices are unique to TAPL. The remaining six helices and the ABC region form the core structure of ABC transporters. The amino‐terminal four helices were revealed to be sufficient for the lysosomal targeting. A truncated protein lacking this region failed in sorting to lysosomes. The latter construct could dimerize, which is essential for half‐type transporters, while the former could not (BBRC, in press). The results demonstrate that TAPL can be dissected into two parts, each required for subcellular localization and transporter machinery.Supported by JSPS grants (13142206, 18590072).

OBTURATOR NERVE INJURY DURING ROBOTIC ASSISTED RADICAL CYSTECTOMY: RECOGNITION OF INJURY AND ROBOTIC REPAIR

Citron-N (citron) binds activated Rho and concentrates at excitatory postsynaptic densities on the smooth dendritic shafts of hippocampal GABAergic neurons. Since little is known about how cytoplasmic proteins become targeted and retained at synapses, we asked how citron attains this discrete distribution. We first sought to determine whether the synaptic localization machinery is unique to GABAergic interneurons, but in neurons cultured from either cerebral cortex or hippocampus, low levels of citron are found in a small population of glutamatergic neurons concentrated at the tips of dendritic spines in addition to shaft synapses. These data suggest that the targeting domains in citron can be utilized appropriately by any neuron, and consistent with this idea, tagged, exogenous, full-length citron becomes concentrated at postsynaptic sites in all hippocampal neurons in which it is introduced. Citron contains multiple sites for protein–protein interaction, including coiled-coil, pleckstrin homology, cysteine-rich zinc binding and CMG domains, as well as a proline-rich region and a PDZ binding tail that is known to interact with PSD-95/SAP90. Using citron deletion mutants, we find that the coiled-coil (CC) domain, located in the N-terminal half of the protein, mediates somatodendritic targeting, self-oligomerization and puncta formation independent of the Rho binding domain, while regions in the C-terminal half of citron are responsible for precise alignment of postsynaptic citron clusters at sites apposing presynaptic boutons. Rho binding and activation regulate membrane targeting and the number of synapses containing citron. Thus, citron's clustering at postsynaptic membranes is mediated by multiple domains, and its synaptic distribution is modified in response to Rho activation.

Cellular Targeting And Function Of Trpc4 Channels In Human Vascular Endothelium

TRPC4 has been suggested as a Ca2+ entry channel, which governs endothelial permeability. In an attempt to identify mechanisms that link TRPC4 function and cell adhesion, we tested the hypothesis that TRPC4 is part of the local signal transduction machinery within adherens junctions. In HEK293 cells transiently co-transfected with VE-Cadherin and TRPC4 constructs, we observed a co-localization of the two proteins within cell-cell contacts. In human microvascular endothelial cells (HMEC), endogenous TRPC4 was found to co-precipitate with two essential components of junctional complexes, VE-cadherin and β-Catenin. Membrane presentation of TRPC4 strongly promoted the formation of cell-cell contact and modified the response to pro-inflammatory stimuli. We observed that both basal- as well as agonist-stimulated Ca2+ influx were substantially augmented by the formation of cell-cell contact in HMEC. Furthermore, we found a significant increase in TRPC4-mediated Ca2+ signals and membrane currents in response to the formation of cell-cell contacts in TRPC4 and VE-cadherin-expressing HEK293 cells. We propose recruitment of TRPC4 proteins into cell-cell contacts as a key mechanism for control of endothelial Ca2+ signalling.

The Role of Agricultural Mechanization in the Modernization of Asian Agriculture: Taiwan's Experience

The demand for mechanization and automation in the agricultural field is a response to the demand for high quality products and sophisticated production techniques in countries with high labor costs. Taiwan started its ten-year mechanization program in 1960 through the introduction of power tillers. Additional measures such as the introduction of field and post-harvest rice drying mechanization, agricultural automation, and precision agriculture were promoted during the past thirty years. Over this period, Taiwan has become a highly mechanized country in rice production. The local small and medium-sized farm machinery industrial sector is characterized by low volume sales of a great variety of farm machinery. Although government policies help to fulfill domestic demand for farm machinery, the local agricultural machinery industry is at a disadvantage as it faces global competition in the new millennium.

Interactions between the juvenile Batten disease gene, CLN3, and the Notch and JNK signalling pathways

Mutations in the gene CLN3 are responsible for the neurodegenerative disorder juvenile neuronal ceroid lipofuscinosis or Batten disease. CLN3 encodes a multi-spanning and hydrophobic transmembrane protein whose function is unclear. As a consequence, the cell biology that underlies the pathology of the disease is not well understood. We have developed a genetic gain-of-function system in Drosophila to identify functional pathways and interactions for CLN3. We have identified previously unknown interactions between CLN3 and the Notch and Jun N-terminal kinase signalling pathways and have uncovered a potential role for the RNA splicing and localization machinery in regulating CLN3 function.

Golgi Localization of Glycosyltransferases Requires a Vps74p Oligomer

The mechanism of glycosyltransferase localization to the Golgi apparatus is a long-standing question in secretory cell biology. All Golgi glycosyltransferases are type II membrane proteins with small cytosolic domains that contribute to Golgi localization. To date, no protein has been identified that recognizes the cytosolic domains of Golgi enzymes and contributes to their localization. Here, we report that yeast Vps74p directly binds to the cytosolic domains of cis and medial Golgi mannosyltransferases and that loss of this interaction correlates with loss of Golgi localization of these enzymes. We have solved the X-ray crystal structure of Vps74p and find that it forms a tetramer, which we also observe in solution. Deletion of a critical structural motif disrupts tetramer formation and results in loss of Vps74p localization and function. Vps74p is highly homologous to the human GMx33 Golgi matrix proteins, suggesting a conserved function for these proteins in the Golgi enzyme localization machinery.

Polo inhibits progenitor self-renewal and regulates Numb asymmetry by phosphorylating Pon

Self-renewal and differentiation are cardinal features of stem cells. Asymmetric cell division provides one fundamental mechanism by which stem cell self-renewal and differentiation are balanced. A failure of this balance could lead to diseases such as cancer. During asymmetric division of stem cells, factors controlling their self-renewal and differentiation are unequally segregated between daughter cells. Numb is one such factor that is segregated to the differentiating daughter cell during the stem-cell-like neuroblast divisions in Drosophila melanogaster, where it inhibits self-renewal. The localization and function of Numb is cell-cycle-dependent. Here we show that Polo (ref. 13), a key cell cycle regulator, the mammalian counterparts of which have been implicated as oncogenes as well as tumour suppressors, acts as a tumour suppressor in the larval brain. Supernumerary neuroblasts are produced at the expense of neurons in polo mutants. Polo directly phosphorylates Partner of Numb (Pon, ref. 16), an adaptor protein for Numb, and this phosphorylation event is important for Pon to localize Numb. In polo mutants, the asymmetric localization of Pon, Numb and atypical protein kinase C are disrupted, whereas other polarity markers are largely unaffected. Overexpression of Numb suppresses neuroblast overproliferation caused by polo mutations, suggesting that Numb has a major role in mediating this effect of Polo. Our results reveal a biochemical link between the cell cycle and the asymmetric protein localization machinery, and indicate that Polo can inhibit progenitor self-renewal by regulating the localization and function of Numb.

Production of sex steroid hormones from DHEA in articular chondrocyte of rats

Dehydroepiandrosterone (DHEA), a precursor of sex steroid hormones, is synthesized by cholesterol side-chain cleavage cytochrome P-450 and 17alpha-hydroxylase cytochrome P-450 mainly from cholesterol and converted to testosterone and estrogen by 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17beta-HSD, and aromatase cytochrome P-450. Although sex steroid hormones have important effects in the protection of articular cartilage, it is unclear whether articular cartilage has a local steroidogenic enzymatic machinery capable of metabolizing DHEA. This study was aimed to clarify whether steroidogenesis-related enzymes are expressed in articular chondrocytes, whether expression levels are changed by DHEA, and whether articular chondrocytes are capable of synthesizing sex steroid hormones from DHEA. Articular chondrocytes isolated from adult rats were cultured with DHEA for 3 days. All of the mRNA expressions of steroidogenesis-related enzymes were detected in cultured articular chondrocytes of rats, but the mRNA expression levels of testosterone and estradiol in cultured media increased after the addition of DHEA. These findings provided the first evidence that articular chondrocytes expressed steroidogenesis-related enzyme genes and that they are capable of locally synthesizing sex steroid hormones locally from DHEA.

Proteomic Analysis of Rat Prefrontal Cortex in Three Phases of Morphine-Induced Conditioned Place Preference

Morphological alterations of synapse are found after morphine administration, suggesting that regulation of synaptic plasticity may be one of the mechanisms of neuroadaptation in addiction. However, the molecular basis underlying the abnormal synapse morphological and physiological changes in the morphine-induced dependence, withdraw, and relapse is not well understood. As prefrontal cortex (PFC) is one of the most important brain regions, which provides executive control over drug use and is severely impaired in many addicts, systematic analysis of the biochemical and molecular alteration of synaptic fraction of PFC in morphine-induced neuroadaptation is necessary. In this study, differential protein expression profiling of synaptic fraction of rat PFC based on morphine-induced conditioned place preference (CPP) model was performed with two-dimensional gel electrophoresis (2-DE). Our results showed that a total of 80 proteins were differentially expressed by 2-DE analysis during three phases of CPP assay. Of them, 58 were further identified by mass spectrometry. These proteins were classified into multiple categories, such as energy metabolism, signal transduction, synaptic transmission, cytoskeletal proteins, chaperones, and local synaptic protein synthetic machinery according to their biological functions. Our study provides a global view of synaptic-related molecular networking in PFC under morphine-induced dependence, withdraw, and relapse, indicative of a concerted biological process in neuroadaptation under chronic morphine exposure.

Expression of steroidogenic enzymes and synthesis of sex steroid hormones from DHEA in skeletal muscle of rats

The functional importance of sex steroid hormones (testosterone and estrogens), derived from extragonadal tissues, has recently gained significant appreciation. Circulating dehydroepiandrosterone (DHEA) is peripherally taken up and converted to testosterone by 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, and testosterone in turn is irreversibly converted to estrogens by aromatase cytochrome P-450 (P450arom). Although sex steroid hormones have been implicated in skeletal muscle regulation and adaptation, it is unclear whether skeletal muscles have a local steroidogenic enzymatic machinery capable of metabolizing circulating DHEA. Thus, here, we investigate whether the three key steroidogenic enzymes (3beta-HSD, 17beta-HSD, and P450arom) are present in the skeletal muscle and are capable of generating sex steroid hormones. Consistent with our hypothesis, the present study demonstrates mRNA and protein expression of these enzymes in the skeletal muscle cells of rats both in vivo and in culture (in vitro). Importantly, we also show an intracellular formation of testosterone and estradiol from DHEA or testosterone in cultured muscle cells in a dose-dependent manner. These findings are novel and important in that they provide the first evidence showing that skeletal muscles are capable of locally synthesizing sex steroid hormones from circulating DHEA or testosterone.

Personal Experience of Replantation for Tamai Zone I Fingertip Amputation

Fingertip amputations in Changhua usually take place because of the intensive distribution of local machinery plants in that area. Though replantation of the fingertip is not a routine procedure because of the cost in operative time and energy to achieve success, the advantages of improved function, nail salvage, elimination of the need for donor sites for later reconstruction and the patient's willingness to maintain bodily integrity meant it has been almost the first choice procedure of our patients in middle Taiwan. 24 Tamai zone I fingertip replantations (amputation at or near lunula) of 22 patients were performed by a single surgeon at Show Chuan Memorial Hospital from December 2002 to October 2004. There were 13 male and 9 female patients, ages ranging from 21 to 59 with an average of 37 years. 18 patients underwent complete amputation and 4 patients had an incomplete amputation without vascular tissue connection. The cases of composite graft were excluded. The type of injury for all patients was crush injury. All patients received microvascular surgery. Artery-only anatomizes with external bleeding via fish mouth incision over the fingertip were performed on 16 digits, and venous drainage was created in the other 8 digits. The replantations were completed under standard microsurgery procedures except for the nerve coaptation and tendon repair. The usual adjuvant medication such as volume expander and anti-thrombin agents were administered for at least 5 days following surgery. Of the 24 digits, 13 digits achieved complete survival, 3 digits partially survived and 8 digits failed to survive. The overall survival rate of the crush injury patients was 66.6%. In successful follow-up cases, all had satisfactory nail regeneration and appearance except one flexion deformity of DIP joint and one nail deviation. The mean DIP flexion was 35.7° and average static two point discrimination was 10.6mm; only one patient experienced cold intolerance. Microsurgical replantation is the only one stage procedure to preserve nails with minor donor site morbidity, especially in crush cases. In this article, we will review the literature and share personal experiences. (J Plast Surg Asso R.O.C. 2006; 15:229~240)

Wisconsin manufacturing is a lot more than just cheese: Surface finishing suppliers thrive from booming local machinery industry

Wisconsin manufacturing is a lot more than just cheese: Surface finishing suppliers thrive from booming local machinery industry