The indispensable role of CCR5 for in vivo suppressor function of tumor-derived CD103+ effector/memory regulatory T cells

CD103 is a marker for identification of effector/memory regulatory T cells (Tregs). CD103(+) Tregs are potent suppressors of tissue inflammation in several infectious diseases, autoimmune diseases, and cancers. However, the underlying mechanisms for this potent suppression ability remain unclear. The current study was designed to clarify this issue. Unexpectedly, we found both CD103(+) and CD103(-) Tregs had similar suppression capacity in vitro. We then chose a murine tumor model for investigation of the in vivo behavior of these Tregs. The suppression ability in vivo against the anti-tumor ability of CD8(+) T cells was restricted to CD103(+) Tregs although both Tregs had equal in vitro suppression ability. In addition, CD103(+) Tregs expressed significantly higher levels of CCR5 than those of CD103(-) Tregs and accumulated more in tumors than did CD103(-) Tregs. Furthermore, blockade of CCR5 signaling, either by CCR5(-/-)CD103(+) Tregs or by CCL5 knockdown tumor, could reduce the migration of CD103(+) Tregs into tumors and impair their in vivo suppression ability. In conclusion, these results indicate that the potent in vivo suppression ability of CD103(+) Tregs is due to the tissue-migration ability through CCR5 expression.     

Characterization and localization of two forms of gonadotropin-releasing hormone (GnRH) in the spinal cord of the frog Rana ridibunda

Two molecular variants of gonadotropin-releasing hormone (GnRH) have been previously characterized in the brain of amphibians, i.e., mammalian GnRH (mGnRH) and chicken GnRH-II (cGnRH-II). The aim of the present study was to identify the molecular forms of gonadotropin-releasing hormone and to localize gonadotropin-releasing hormone-containing elements in the spinal cord of the frog Rana ridibunda using highly specific antisera against mGnRH and cGnRH-II. High-performance liquid chromatography (HPLC) analysis combined with radioimmunoassay (RIA) detection revealed that frog spinal cord extracts contained both mGnRH and cGnRH-II. Immunohistochemical labeling revealed that the frog spinal cord was devoid of GnRH-containing cell bodies. In contrast, numerous GnRH-immunoreactive fibers were observed throughout the entire length of the cord. mGnRH immunoreactivity was only detected in the rostral region of the cord and consisted of varicose processes located in the vicinity of the central canal. cGnRH-II-positive fibers were found throughout the spinal cord, the density of immunoreactive processes decreasing gradually toward the caudal region. Two main cGnRH-II-positive fiber tracts with a rostrocaudal orientation were observed: a relatively dense fiber bundle surrounding the central canal, and a more diffuse plexus in the white matter. In addition, short, varicose cGnRH-II-positive processes with a radial orientation were present throughout the gray matter. These fibers were particularly abundant ventromedially and formed a diffuse network that ramified laterally to end in the vicinity of motoneurons. Taken together, these data indicate that the frog spinal cord, like the frog brain, contains two forms of GnRH. The presence of numerous cGnRH-II-immunoreactive fibers in the ventral horn suggests that cGnRH-II may influence the activity of a subpopulation of motoneurons.     

Changes in mitochondrial DNA levels during early embryogenesis in Torenia fournieri and Arabidopsis thaliana

Changes in the amount of mitochondrial DNA (mtDNA) have never been investigated in plant zygotes or early plant embryos due to the difficulty in isolating these cells, although such changes have been investigated in mammalian embryos. Using the single‐cell quantitative real‐time polymerase chain reaction (PCR) and laser confocal microscopy, we surveyed the changes in mtDNA levels during early embryogenesis in Torenia fournieri and Arabidopsis thaliana. In contrast with the amount of mtDNA in early mammalian embryos, which does not change, we found that mtDNA doubling occurred during zygotic development in T. fournieri and during two‐cell proembryo development in A. thaliana. These findings reveal that mtDNA doubling occurs during early embryogenesis in T. fournieri and A. thaliana, indicating that the dynamics of mtDNA in early plant embryos differs from that in early mammalian embryos.     

Berberine Targets AP-2/hTERT,NF-κB/COX-2, HIF-1α/VEGF and Cytochrome-c/Caspase Signaling to Suppress Human Cancer Cell Growth

Berberine (BBR), an isoquinoline derivative alkaloid isolated from Chinese herbs, has a long history of uses for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of BBR in human lung cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which BBR inhibits cell growth in human non-small-cell lung cancer (NSCLC) cells. Treatment with BBR promoted cell morphology change, inhibited cell migration, proliferation and colony formation, and induced cell apoptosis. Further molecular mechanism study showed that BBR simultaneously targeted multiple cell signaling pathways to inhibit NSCLC cell growth. Treatment with BBR inhibited AP-2α and AP-2β expression and abrogated their binding on hTERT promoters, thereby inhibiting hTERT expression. Knockdown of AP-2α and AP-2β by siRNA considerably augmented the BBR-mediated inhibition of cell growth. BBR also suppressed the nuclear translocation of p50/p65 NF-κB proteins and their binding to COX-2 promoter, causing inhibition of COX-2. BBR also downregulated HIF-1α and VEGF expression and inhibited Akt and ERK phosphorylation. Knockdown of HIF-1α by siRNA considerably augmented the BBR-mediated inhibition of cell growth. Moreover, BBR treatment triggered cytochrome-c release from mitochondrial inter-membrane space into cytosol, promoted cleavage of caspase and PARP, and affected expression of BAX and Bcl-2, thereby activating apoptotic pathway. Taken together, these results demonstrated that BBR inhibited NSCLC cell growth by simultaneously targeting AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK and cytochrome-c/caspase signaling pathways. Our findings provide new insights into understanding the anticancer mechanisms of BBR in human lung cancer therapy.     

An-1 Encodes a Basic Helix-Loop-Helix Protein That Regulates Awn Development,Grain Size,and Grain Number in Rice

Long awns are important for seed dispersal in wild rice (Oryza rufipogon), but are absent in cultivated rice (Oryza sativa). The genetic mechanism involved in loss-of-awn in cultivated rice remains unknown. We report here the molecular cloning of a major quantitative trait locus, An-1, which regulates long awn formation in O. rufipogon. An-1 encodes a basic helix-loop-helix protein, which regulates cell division. The nearly-isogenic line (NIL-An-1) carrying a wild allele An-1 in the genetic background of the awnless indica Guangluai4 produces long awns and longer grains, but significantly fewer grains per panicle compared with Guangluai4. Transgenic studies confirmed that An-1 positively regulates awn elongation, but negatively regulates grain number per panicle. Genetic variations in the An-1 locus were found to be associated with awn loss in cultivated rice. Population genetic analysis of wild and cultivated rice showed a significant reduction in nucleotide diversity of the An-1 locus in rice cultivars, suggesting that the An-1 locus was a major target for artificial selection. Thus, we propose that awn loss was favored and strongly selected by humans, as genetic variations at the An-1 locus that cause awn loss would increase grain numbers and subsequently improve grain yield in cultivated rice.     

Efficient production of S-adenosyl-l-methionine from dl-methionine in metabolic engineered Saccharomyces cerevisiae

S-Adenosyl-l -methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although l -methionine is generally used, the low-cost dl -methionine is more suitable as the substrate for industrial production of SAM. However, d -methionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of dl -methionine, intracellular conversion of d -methionine to l -methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding d -amino acid-N-acetyltransferase, d -methionine was accumulated in vivo and no N-acetyl-d -methionine production was observed. Further, codon-optimized d -amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and l -phenylalanine dehydrogenase gene (l -PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert d -methionine to l -methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L d -methionine. Using the recently developed CRISPR tools, the DAAO and l -PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L dl -methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L dl -methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost dl -methionine.     

On signaling pathways: hematopoietic stem cell specification from hemogenic endothelium

Hematopoietic stem cells(HSCs) are specified and generated during the embryonic development and have remarkable potential to replenish the full set of blood cell lineages. Researchers have long been interested in clarifying the molecular events involved in HSC specification. Many studies have reported the development of methods for generating functional hematopoietic cells from pluripotent stem cells(PSCs-embryonic stem cells(ESCs) and induced pluripotent stem cells(i PSCs)) for decades. However, the generation of HSCs with robust long-term repopulation potential remains a swingeing challenge, of which a major factor contributing to this failure is the difficulty to define the intraembryonic signals related to the specification of HSCs. Since HSCs directly derive from hemogenic endothelium, in this review, we summarize both in vivo and in vitro studies on conserved signaling pathways that control the specification of HSCs from hemogenic endothelial cells.