Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.     

Pain modality and spinal glia expression by streptozotocin induced diabetic peripheral neuropathy in rats

Pain symptoms are a common complication of diabetic peripheral neuropathy or an inflammatory condition. In the most experiments, only one or two evident pain modalities are observed at diabetic peripheral neuropathy according to experimental conditions. Following diabetic peripheral neuropathy or inflammation, spinal glial activation may be considered as an important mediator in the development of pain. For this reason, the present study was aimed to address the induction of pain modalities and spinal glial expression after streptozotocin injection as compared with that of zymosan inflammation in the rat. Evaluation of pain behavior by either thermal or mechanical stimuli was performed at 3 weeks or 5 hours after either intravenous streptozotocin or zymosan. Degrees of pain were divided into 4 groups: severe, moderate, mild, and non-pain induction. On the mechanical allodynia test, zymosan evoked predominantly a severe type of pain, whereas streptozotocin induced a weak degree of pain (severe+moderate: 57.1%). Although zymosan did not evoke cold allodynia, streptozotocin evoked stronger pain behavior, compared with zymosan (severe+moderate: 50.0%). On the other hand, the high incidence of thermal hyperalgesia (severe+moderate: 90.0%) and mechanical hyperalgesia (severe+moderate: 85.7%) by streptozotocin was observed, as similar to that of zymosan. In the spinal cord, the increase of microglia and astrocyte was evident by streptozotocin, only microglia was activated by zymosan. Therefore, it is recommended that the selection of mechanical and thermal hyperalgesia is suitable for the evaluation of streptozotocin induced diabetic peripheral neuropathy. Moreover, spinal glial activation may be considered an important factor.     

Progression of Parkinson's Disease Pathology Is Reproduced by Intragastric Administration of Rotenone in Mice

In patients with Parkinson''s disease (PD), the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS), the dorsal motor nucleus of the vagus (DMV), the intermediolateral nucleus of the spinal cord and the substantia nigra, providing the basis for the neuropathological staging of the disease. Here we report that intragastrically administered rotenone, a commonly used pesticide that inhibits Complex I of the mitochondrial respiratory chain, is able to reproduce PD pathological staging as found in patients. Our results show that low doses of chronically and intragastrically administered rotenone induce alpha-synuclein accumulation in all the above-mentioned nervous system structures of wild-type mice. Moreover, we also observed inflammation and alpha-synuclein phosphorylation in the ENS and DMV. HPLC analysis showed no rotenone levels in the systemic blood or the central nervous system (detection limit [rotenone]<20 nM) and mitochondrial Complex I measurements showed no systemic Complex I inhibition after 1.5 months of treatment. These alterations are sequential, appearing only in synaptically connected nervous structures, treatment time-dependent and accompanied by inflammatory signs and motor dysfunctions. These results strongly suggest that the local effect of pesticides on the ENS might be sufficient to induce PD-like progression and to reproduce the neuroanatomical and neurochemical features of PD staging. It provides new insight into how environmental factors could trigger PD and suggests a transsynaptic mechanism by which PD might spread throughout the central nervous system.     

DHP-Derivative and Low Oxygen Tension Effectively Induces Human Adipose Stromal Cell Reprogramming

Background and Methods

In this study, we utilized a combination of low oxygen tension and a novel anti-oxidant, 4-(3,4-dihydroxy-phenyl)-derivative (DHP-d) to directly induce adipose tissue stromal cells (ATSC) to de-differentiate into more primitive stem cells. De-differentiated ATSCs was overexpress stemness genes, Rex-1, Oct-4, Sox-2, and Nanog. Additionally, demethylation of the regulatory regions of Rex-1, stemnesses, and HIF1α and scavenging of reactive oxygen species were finally resulted in an improved stem cell behavior of de-differentiate ATSC (de-ATSC). Proliferation activity of ATSCs after dedifferentiation was induced by REX1, Oct4, and JAK/STAT3 directly or indirectly. De-ATSCs showed increased migration activity that mediated by P38/JUNK and ERK phosphorylation. Moreover, regenerative efficacy of de-ATSC engrafted spinal cord-injured rats and chemical-induced diabetes animals were significantly restored their functions.

Conclusions/Significance

Our stem cell remodeling system may provide a good model which would provide insight into the molecular mechanisms underlying ATSC proliferation and transdifferentiation. Also, these multipotent stem cells can be harvested may provide us with a valuable reservoir of primitive and autologous stem cells for use in a broad spectrum of regenerative cell-based disease therapy.     

Localized Populations of CD8low/? MHC Class I Tetramer+ SIV-Specific T Cells in Lymphoid Follicles and Genital Epithelium

CD8 T cells play an important role in controlling viral infections. We investigated the in situ localization of simian immunodeficiency virus (SIV)-specific T cells in lymph and genital tissues from SIV-infected macaques using MHC-class I tetramers. The majority of tetramer-binding cells localized in T cell zones and were CD8⁺. Curiously, small subpopulations of tetramer-binding cells that had little to no surface CD8 were detected in situ both early and late post-infection, and in both vaginally and rectally inoculated macaques. These tetramer⁺CD8^low/− cells were more often localized in apparent B cell follicles relative to T cell zones and more often found near or within the genital epithelium than the submucosa. Cells analyzed by flow cytometry showed similar populations of cells. Further immunohistological characterization revealed small populations of tetramer⁺CD20⁻ cells inside B cell follicles and that tetramer⁺ cells did not stain with γδ-TCR nor CD4 antibodies. Negative control tetramer staining indicated that tetramer⁺CD8^low/− cells were not likely NK cells non-specifically binding to MHC tetramers. These findings have important implications for SIV-specific and other antigen-specific T cell function in these specific tissue locations, and suggest a model in which antigen-specific CD8+ T cells down modulate CD8 upon entering B cell follicles or the epithelial layer of tissues, or alternatively a model in which only antigen-specific CD8 T cells that down-modulate CD8 can enter B cell follicles or the epithelium.     

Bicyclic α,ω-dicarboxylic acid derivatives from a colonial tunicate of the family Polyclinidae

In the course of our search for bioactive metabolites from a colonial tunicate of the family Polyclinidae, six new (1–6) cyclic fatty acid derivatives were isolated. Their planar structures were established on the basis of NMR and MS spectroscopic analyses. The relative configuration was determined by NOESY experiment. Compounds 1–6 represent a fused bicyclic skeleton possibly derived from α,ω-dicarboxylic acids such as eicosanedioic acid or docosanedioic acid via a Diels–Alder type of cyclization. Compounds 1–4 and 6 showed mild cytotoxicity against a panel of five human solid tumor cell lines.     

An Arabidopsis F-box protein regulates tapetum degeneration and pollen maturation during anther development

The Arabidopsis anther has a bilateral symmetry with four lobes, each consisting of four distinct layers of somatic cells from the outer to inner side: epidermis, endothecium, middle layer and tapetum. The tapetum is a layer of cells comprising the inner surface of the pollen wall. It plays an important role in anther development by providing enzymes, materials and nutrients required for pollen maturation. Genes and molecular mechanisms underlying tapetum formation and pollen wall biosynthesis have been studied in Arabidopsis. However, tapetum degeneration and anther dehiscence have not been well characterized at the molecular level. Here, we report that an Arabidopsis gene, designated reduced male fertility (RMF), regulates degeneration of tapetum and middle layer during anther development. The Arabidopsis dominant mutant rmf-1D overexpressing the RMF gene exhibited pleiotropic phenotypes, including dwarfed growth with small, dark-green leaves and low male fertility. Tapetum development and subsequent degeneration were impaired in the mutant. Accordingly, pollen maturation was disturbed, reducing the male fertility. In contrast, tapetum degeneration was somewhat accelerated in the RMF RNAi plants. The RMF gene was expressed predominantly in the anther, particularly in the pollen grains. Notably, the RMF protein contains an F-box motif and is localized to the nucleus. It physically interacts with the Arabidopsis-Skp1-like1 protein via the F-box motif. These observations indicate that the RMF gene encodes an F-box protein functioning in tapetum degeneration during anther development.     

Egr-1, a new downstream molecule of Epstein-Barr virus latent membrane protein 1

To investigate the effect of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on human cancer cells, we sought to identify and analyze potential target genes that were differentially expressed in the presence and absence of LMP1. Our cDNA microarray analysis revealed that expression of early growth response gene-1 (Egr-1) was increased by LMP1 expression in MCF7 and Jurkat cells. An NFkappaB inhibitor (SN50) antagonized LMP1-induced enhancement of Egr-1 expression, indicating that LMP1 induced Egr-1 via NFkappaB. Furthermore, three lines of evidence indicated that Egr-1 was required for LMP1-induced cancer cell survival. First, Egr-1 expression enhanced the survival of doxorubicin-treated MCF7 cells. Second, inhibition of Egr-1 expression by siRNA (siEgr-1) effectively suppressed LMP-1-induced survival of MCF7 cells. Third, Egr-1 knockdown decreased LMP1-induced expression of Bfl-1. Similar relationships among EBV infection, Egr-1 and drug resistance were also observed in tissues of peripheral T-cell lymphoma-unspecified (PTCL-u) patients.