Functional characterization of the neuron-restrictive silencer element in the human tryptophan hydroxylase 2 gene expression.

Tryptophan hydroxylase 2 (TPH2) is the key enzyme in the synthesis of neuronal serotonin. Although previous studies suggest that TPH2 neuron-restrictive silencer element (NRSE) functions as a negative regulator dependent on neuron-restrictive silencer factor (NRSF) activity, the underlying mechanisms are yet to be fully elucidated. Here, we show a detailed analysis of the NRSE-mediated repression of the human TPH2 (hTPH2) promoter activity in RN46A cells, a cell line derived from rat raphe neurons. Quantitative real-time RT-PCR analysis revealed the expression of serotonergic marker genes (Mash1, Nkx2.2, Gata2, Gata3, Lmx1b, Pet-1, 5-Htt, and Vmat2) and Nrsf gene in RN46A cells. Tph1 mRNA is the prevalent form expressed in RN46A cells; Tph2 mRNA is also expressed but at a lower level. Electrophoretic mobility shift assays and reporter assays showed that hTPH2 NRSE is necessary for the efficient DNA binding of NRSF and for the NRSF-dependent repression of the hTPH2 promoter activity. The hTPH2 promoter activity was increased by knockdown of NRSF, or over-expression of the engineered NRSF (a dominant-negative mutant or a DNA-binding domain and activation domain fusion protein). MS-275, a class I histone deacetylase (HDAC) inhibitor, was found to be more potent than MC-1568, a class II HDAC inhibitor, in enhancing the hTPH2 promoter activity. Furthermore, treatment with the ubiquitin-specific protease 7 deubiquitinase inhibitors, P-22077 or HBX 41108, increased the hTPH2 promoter activity. Collectively, our data demonstrate that the hTPH2 NRSE-mediated promoter repression via NRSF involves class I HDACs and is modulated by the ubiquitin-specific protease 7-mediated deubiquitination and stabilization of NRSF.

l-Leucylglycylglycine.

In the title compound, C10H19N3O4, the N- and C-termini are protonated and ionized, respectively, and the mol-ecule forms a zwitterion. The main chain is in a folded form. In the crystal, the N-terminal -NH3 (+) group hydrogen bonds to three C-terminal -COO groups and one carbonyl O atom, forming a three-dimensional network. In addition, an N-H⋯O hydrogen bond between the amide groups of the middle glycine residue and a C-H⋯O inter-action continue along the a-axis direction. The side chains of the leucyl residues form a hydro-phobic region along the a axis.

Simple method for large-scale production of macrophage activating factor GcMAF.

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine420 (Thr420) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr420. In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing.

Oxidized galectin-1 stimulates macrophages to promote axonal regeneration in peripheral nerves after axotomy.

Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt neurons to send out processes in peripheral nerves after axotomy are not well understood. Previously, we have shown oxidized galectin-1 (GAL-1/Ox) promotes initial axonal growth after axotomy in peripheral nerves. However, the mechanism by which GAL-1/Ox promotes axonal regeneration remains unclear and is the subject of the present study. To identify possible target cells of GAL-1/Ox, a fluorescently labeled recombinant human GAL-1/Ox (rhGAL-1/Ox) was incubated with DRG neurons, Schwann cells, and intraperitoneal macrophages from adult rats. Only the cell surfaces of intraperitoneal macrophages bound the rhGAL-1/Ox, suggesting that these cells possess a receptor for GAL-1/Ox. Experiments examining tyrosine phosphorylation revealed that rhGAL-1/Ox stimulated changes in signal transduction pathways in these macrophages. These changes caused macrophages to secrete an axonal growth-promoting factor. This was demonstrated when conditioned media of macrophages stimulated with rhGAL-1/Ox in 48 hr culture strongly enhanced axonal regeneration from transected-nerve sites of DRG explants. Furthermore, activated macrophage-conditioned media also improved Schwann cell migration from the transected-nerve sites. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced galectin-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. Oxidized galectin-1 then stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration.

Nucleotide sequence for cDNA of bovine mitochondrial ATP-dependent protease and determination of N-terminus of the mature enzyme from the adrenal cortex.

We have determined the cDNA sequence encoding bovine mitochondrial ATP-dependent Lon protease. Since the 5'-end region of the cDNA was highly GC-rich and thus could not be amplified by the 5'-RACE method, a genomic DNA fragment containing an in-frame ATG was isolated and sequenced. The translated amino acid sequence contained 961 amino acids with a calculated molecular weight 106,665. Sequence similarities of the bovine enzyme to human and E. coli orthologs were 92 and 27%, respectively. The N-terminal amino acid sequence seemed to be a mitochondrial targeting signal. To determine the cleavage site of the signal sequence we analyzed the mature enzyme purified from bovine adrenocortical mitochondria. Analysis of CNBr-digested peptides revealed that the N-terminus was heterogeneous. We suggest that nonspecific aminopeptidase might remove several amino acids from the N-terminus after mitochondrial processing peptidase has cleaved Gly(67)-Leu(68) or Leu(68)-Trp(69).