Axonal-Transport-Mediated Gene Transduction in the Interior of Rat Bone

Background

Gene transduction has been considered advantageous for the sustained delivery of proteins to specific target tissues. However, in the case of hard tissues, such as bone, local gene delivery remains problematic owing to anatomical accessibility limitations of the target sites.

Methodology/Principal Findings

Here, we evaluated the feasibility of exogenous gene transduction in the interior of bone via axonal transport following intramuscular administration of a nonviral vector. A high expression level of the transduced gene was achieved in the tibia ipsilateral to the injected tibialis anterior muscle, as well as in the ipsilateral sciatic nerve and dorsal root ganglia. In sciatic transection rats, the gene expression level was significantly lowered in bone.

Conclusions/Significance

These results suggest that axonal transport is critical for gene transduction. Our study may provide a basis for developing therapeutic methods for efficient gene delivery into hard tissues.     

Diurnal changes of ERP response to sound stimuli of varying frequency in morning-type and evening-type subjects

In order to study the cognitive function rhythm related to the auditory frequency system for people who prefer to be active in the morning and at night, we conducted an experiment during morning (09:00), evening (17:00) and late-night (01:00) periods. On the basis of a morningness/eveningness questionnaire, six moderately morning-type subjects (M-types) and seven evening-type subjects (E-types) were selected. Diurnal variation of event-related potential (ERP) were assessed under low-frequency (250/500 Hz) and high-frequency (1000/2000 Hz) condition using an oddball task. M-types were tested during the morning (09:00) and evening (17:00) periods, and E-types were tested during the evening (17:00) and midnight (01:00) periods. Subjects were asked to press a button when the target stimulus was detected. We found that the P300 amplitude at 09:00 was significantly greater than that at 17:00 for M-types, was significantly greater at 17:00 than that at 01:00 for E-types. A significant difference of P300 latency and P300 amplitude was observed at 17:00 between M-types and E-types. The P300 amplitude obtained after a low-frequency stimulus was significantly greater than that after a high-frequency stimulus at 09:00 for M-types, and at 01:00 for E-types. These results revealed that stimulus frequency had effects on the diurnal changes of human cognitive function, and circadian typology had a direct effect on the diurnal change of human cognitive function. This study has extended the previous findings of auditory P300 studies on diurnal variations in terms of circadian typology and stimulus parameter.     

Migration of zebrafish primordial germ cells: a role for myosin contraction and cytoplasmic flow

The molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration.     

Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis

Bone homeostasis requires a delicate balance between the activities of bone-resorbing osteoclasts and bone-forming osteoblasts. Various molecules coordinate osteoclast function with that of osteoblasts; however, molecules that mediate osteoclast-osteoblast interactions by simultaneous signal transduction in both cell types have not yet been identified. Here we show that osteoclasts express the NFATc1 target gene Efnb2 (encoding ephrinB2), while osteoblasts express the receptor EphB4, along with other ephrin-Eph family members. Using gain- and loss-of-function experiments, we demonstrate that reverse signaling through ephrinB2 into osteoclast precursors suppresses osteoclast differentiation by inhibiting the osteoclastogenic c-Fos-NFATc1 cascade. In addition, forward signaling through EphB4 into osteoblasts enhances osteogenic differentiation, and overexpression of EphB4 in osteoblasts increases bone mass in transgenic mice. These data demonstrate that ephrin-Eph bidirectional signaling links two major molecular mechanisms for cell differentiation--one in osteoclasts and the other in osteoblasts--thereby maintaining bone homeostasis.     

Complex formation of Plk1 and INCENP required for metaphase-anaphase transition

Mitotic chromosomal dynamics is regulated by the coordinated activities of many mitotic kinases, such as cyclin-dependent kinase 1 (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but the mechanisms of their coordination remain unknown. Here, we report that Cdk1 phosphorylates Thr 59 and Thr 388 on inner centromere protein (INCENP), which regulates the localization and kinase activity of Aurora-B from prophase to metaphase. INCENP depletion disrupts Plk1 localization specifically at the kinetochore. This phenotype is rescued by the exogenous expression of INCENP wild type and INCENP mutated at Thr 59 to Ala (T59A), but not at Thr 388 to Ala (T388A). The replacement of endogenous INCENP with T388A resulted in the delay of progression from metaphase to anaphase. We propose that INCENP phosphorylation by Cdk1 is necessary for the recruitment of Plk1 to the kinetochore, and that the complex formation of Plk1 and Aurora-B on INCENP may play crucial roles in the regulation of chromosomal dynamics.     

G-CSF-induced evacuation of sinusoidal NK cells and the facilitation of liver regeneration in a partial hepatectomy

Since liver regeneration after partial hepatectomy (PHx) is known to improve by pretreatment with recombinant human G-CSF (rhG-CSF), we investigated the mechanism by evaluating the distribution and activity of sinusoidal NK cells. F344 rats were treated with rhG-CSF (250 microg/kg/day) for 5 days before PHx. Pretreatment with rhG-CSF improved the serum ALT levels and DNA biosynthesis of the remnant liver tissues at 20 h after PHx. Notably, the rhG-CSF pretreatment decreased the number of NK cells in the liver determined by immunohistochemistry using anti-NKR-P1A mAb before and at 20 h after PHx with no significant change in the NK activity per cell base, while also increasing the number of NK cells in the peripheral blood detected by flow cytometry. The rhG-CSF induced a pre-PHx downregulation of the IL-12p70 protein levels, while also promoting the post-PHx reduction of the protein levels of IL-12p70 and IFN-gamma. Conversely, rhG-CSF had no effect on the pre-PHx mRNA levels or the PHx-induced upregulation of mRNA levels of TNF-alpha, IL-1beta, IL-6, TGF-beta, IL-10, HGF, and c-Met determined by real-time RT-PCR. These results strongly suggest that rhG-CSF-induced facilitation of liver regeneration is achieved by immunoregulation through the intrahepatic IL-12 downregulation and evacuation of sinusoidal NK cells.     

The in vitro real-time oscillation monitoring system identifies potential entrainment factors for circadian clocks

Background  

Circadian rhythms are endogenous, self-sustained oscillations with approximately 24-hr rhythmicity that are manifested in various physiological and metabolic processes. The circadian organization of these processes in mammals is governed by the master oscillator within the suprachiasmatic nuclei (SCN) of the hypothalamus. Recent findings revealed that circadian oscillators exist in most organs, tissues, and even in immortalized cells, and that the oscillators in peripheral tissues are likely to be coordinated by SCN, the master oscillator. Some candidates for endogenous entrainment factors have sporadically been reported, however, their details remain mainly obscure.     

Tol2 transposon-mediated transgenesis in Xenopus tropicalis

The diploid frog Xenopus tropicalis is becoming a powerful developmental genetic model system. Sequencing of the X. tropicalis genome is nearing completion and several labs are embarking on mutagenesis screens. We are interested in developing insertional mutagenesis strategies in X. tropicalis. Transposon-mediated insertional mutagenesis, once used exclusively in plants and invertebrate systems, is now more widely applicable to vertebrates. The first step in developing transposons as tools for mutagenesis is to demonstrate that these mobile elements function efficiently in the target organism. Here, we show that the Medaka fish transposon, Tol2, is able to stably integrate into the X. tropicalis genome and will serve as a powerful tool for insertional mutagenesis strategies in the frog.     

UV-irradiation induces oxidative damage to mitochondrial DNA primarily through hydrogen peroxide: analysis of 8-oxodGuo by HPLC

Roles of reactive oxygen species (ROS) in damage to mitochondrial DNA (mtDNA) following ultraviolet (UV)-irradiation were investigated in the human hepatoma cell line SK-HEP-1. We altered the intracellular status of ROS by the overexpression of manganese superoxide dismutase (MnSOD) and/or catalase. Using HPLC, we analyzed 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), known as a marker of damage to DNA molecules. UV-irradiation resulted in the accumulation of 8-oxodGuo in these cells. The overexpression of MnSOD enhanced the accumulation of 8-oxodGuo by UV. The co-overexpression of catalase inhibited the accumulation of 8-oxodGuo by UV in MnSOD-transfectants. The overexpression of MnSOD reduced the colony forming capacity in SK-HEP-1 cells and the co-overexpression of catalase with MnSOD stimulated the capacity compared to control. UV-irradiation inhibited the colony forming capacity in these cells; no difference was observed among the capacities of control, MnSOD- and catalase-transfectants. However, the overexpression of MnSOD/catalase significantly rescued the reduction of colony forming capacity by UV-irradiation. Our results suggest that the accumulation of hydrogen peroxide plays a key role in the oxidative damage to mtDNA of UV-irradiated cells, and also that the overexpression of both MnSOD and catalase reduces the mtDNA damage and blocks the growth inhibition by UV. Our results also indicate that the increased activity of MnSOD may lead to a toxic effect on mtDNA by UV-irradiation.     

Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.