Loss of RNA binding protein HuD facilitates the production of the senescence-associated secretory phenotype

HuD, an RNA binding protein, plays a role in the regulation of gene expression in certain types of cells, including neuronal cells and pancreatic β-cells, via RNA metabolism. Its aberrant expression is associated with the pathogenesis of several human diseases. To explore HuD-mediated gene regulation, stable cells expressing short hairpin RNA against HuD were established using mouse neuroblastoma Neuro2a (N2a) cells, which displayed enhanced phenotypic characteristics of cellular senescence. Two approaches, RNA immunoprecipitation (RNA IP)-NanoString profiling and cytokine array, were used to subsequently identify a subset of putative HuD targets that act as senescence-associated secretory phenotype (SASP), including C-C motif ligand 2 (CCL2), CCL20, C-X-C motif chemokine ligand 2 (CXCL2), and interleukin-6 (IL-6). Here, we further demonstrated that HuD regulates the expression of CCL2, a SASP candidate upregulated in cells following HuD knockdown, by binding to the 3′-untranslated region (UTR) of Ccl2 mRNA. Downregulation of HuD increased the level of CCL2 in N2a cells and the brain tissues of HuD knockout (KO) mice. Exposure to γ-irradiation induced cellular senescence in N2a cells and HuD knockdown facilitated stress-induced cellular senescence. Our results reveal that HuD acts as a novel regulator of CCL2 expression, and its aberrant expression may contribute to cellular senescence by regulating SASP production.Subject terms: Senescence, RNA quality control     

Enzymatic amplification and characterization of large DNA fragments from genomic DNA

Conditions for DNA amplification in vitro using modified T7 DNA polymerase have been devised to obtain 2000-bp DNA fragments of the HGPRT gene directly from human genomic DNA. The DNA obtained from a 1.2 x 10(5)-fold amplification has been used for direct sequencing.     

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.     

Reconstruction of soft-tissue defects using serratus anterior adipofascial free flap

The serratus anterior muscle has been suggested as a versatile and reliable flap for reconstruction of head and neck and extremity injuries. The adipofascial layer overlying the serratus anterior muscle is the anatomic layer, which is supplied by the same branch of thoracodorsal artery. Even though great progress has occurred in the prevention of postoperative adhesion of extremity injuries, the problem has not been completely solved and is still of special importance in complex injuries. Between March of 1995 and February of 1996, seven patients underwent reconstructive operation as a result of soft-tissue defects of the upper or lower extremities or the scalp. We transferred free adipofascial tissue overlying the serratus anterior muscle in three patients and both serratus anterior muscle and adipofascial tissue in four patients. A free adipofascial flap overlying serratus anterior muscle was transferred when a gliding surface was required, owing to the exposure of tendons and neurovascular structures. The average duration from operation to follow-up examination was 8 months (from 4 to 16 months). The results of the operations were satisfactory in functional and cosmetic aspects. This kind of flap was very effective in reconstruction of soft-tissue defects and gliding surfaces for these reasons: easy dissection, the capability of obtaining a long vascular pedicle, large-sized flap, composite flap including muscle or rib, and the fact that there was no serious functional or cosmetic deficit at the donor site.     

WNT5A signaling affects pituitary gland shape

Wnt signaling is important in organogenesis, and aberrant signaling in mature cells is associated with tumorigenesis. Several members of the Wnt family of signaling molecules are expressed in the developing pituitary gland. Wnt5a is expressed in the neuroectoderm that induces pituitary gland development and has been proposed to influence pituitary cell specification. We discovered that mice deficient in Wnt5a display abnormal morphology in the dorsal part of the developing pituitary. The expression of downstream effectors of the canonical Wnt pathway is not altered, and expression of genes in other signaling pathways such as Shh, Fgf8, Fgf10 and Fgfr2b is intact. Prop1 and Hesx1 are also important for normal shape of the pituitary primordium, but their expression is unaltered in the Wnt5a mutants. Specification of the hormone-producing cell types of the mature anterior pituitary gland occurs appropriately. This study suggests that the primary role of Wnt5a in the developing pituitary gland is in establishment of the shape of the gland.     

Biological activity in traditional Alaska pollack sikhae during low temperature fermentation

Biological activity was examined on Alaska pollack sikhae produced with 4 treatments (by irradiating at 5 or 10 kGy, or by adding either 0.1 or 0.3% of chitooligosaccharide), compared with control (2-step fermentation only) during fermentation at -2 degrees C. The extracts (500 ppm level) of sikhae had antimicrobial activities against 4 different strains of food poisoning bacteria such as Staphy. aureus, B. subtilis, B. cereus, and L. monocytogenes. Antioxidative activity (EDA(50), 11.55 mg/mL) in control group increased with time up to 60 days of fermentation but decreased thereafter, while those levels in other products were kept within 10.60-18.30 mg/mL ranges during fermentation. Inhibitory activity of angiotensin-I converting enzyme (ACE) (IC(50), 1.51-2.89 mg/mL) in all products was observed during fermentation except at 0 day. Inhibitory activity of xanthine oxidase (XO) (IC(50), 0.65-0.87 mg/mL) in all products also increased with time up to 30 days of fermentation. Without irradiating or adding of chitooligosaccharide, Alaska pollack sikhae showing biological activities was enough by 2-step fermentation and storage at -2 degrees C only.     

Promoting effect of amino acids added to a chemically defined medium on blastocyst formation and blastomere proliferation of bovine embryos cultured in vitro

This study was conducted to elucidate the role of amino acids added singly or in groups to a chemically defined culture medium in blastocyst formation and blastomere proliferation of bovine embryos. Embryos were generated by in vitro fertilization, and blastocyst formation and hatching, and blastomere number of blastocysts were subsequently monitored after the culture of embryos in synthetic oviduct fluid medium (SOFM). First, one of four non-essential amino acids (asparagine, aspartate, glutamate or serine) was added to SOFM and, compared with no addition, a significant (P <0.05) increase in blastocyst formation was found after the addition of asparagine, aspartate, or glutamate (35-42% versus 22%). Second, one of four essential amino acids (arginine, cystine, isoleucine or leucine) was added and arginine or isoleucine greatly improved blastocyst formation (30-36% versus 16%). Third, the addition of five stimulatory amino acids (aspartate, asparagine, glutamate, arginine and isoleucine) to SOFM significantly improved blastocyst formation compared with no addition (12% versus 21%) and such value was similar to that obtained after the addition of 19 amino acids consisting of MEM amino acid solutions (21-27%). However, five amino acids yielded fewer hatched blastocysts than 19 amino acids. Finally, although five amino acids yielded more cell number of blastocysts than no addition (93 versus 74 cells per blastocyst), it was lower than that from 19 amino acids (131 cells per blastocyst). In conclusion, either single or combined addition of asparagine, aspartate, glutamate, arginine and isoleucine stimulated blastocyst formation, while other amino acids might be necessary for further stimulating blastomere proliferation and blastocyst hatching.     

Enhanced immunodetection of cell-free synthesized erythropoietin under denaturing conditions

A monoclonal antibody produced by hydridoma cell line, ATCC HB8209, was used to detect and purify erythropoietin synthesized in a cell-free system. The antibody was raised against the N-terminal 20 residues of erythropoietin. It retained anti-erythropoietin activity in 6 M urea in which most of the cell-free synthesized erythropoietin became soluble and gave an enhanced activity of the antibody.