In vitro biological activities of glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminic acid

In the previous study, N-acetylneuraminic acid (NANA) with C9 spacer was chemically coupled to human recombinant (rh) IL-1 in order to study the effect of glycosylation on its biological activities, and to develop IL-1 with less deleterious effects. In this study we examined a variety of IL-1 activities in vitro, including proliferative effect on T cells, antiproliferative effect on myeloid leukemic cells and melanoma cells, stimulatory effects on IL-6 synthesis by melanoma cells and PGE₂ synthesis by fibroblast cells. NANA-introduced IL-1 (NANA-IL-1) exhibited reduced activities about ten times compared with original IL-1 in all the activities performed in vitro. The competitive binding of ¹²⁵I-IL-1 to mouse T cells and pre-B cells with unlabeled IL-1s suggests the decrease in binding affinities of NANA-IL-1 to both type I and type II IL-1 receptors. Therefore, reduced activities of NANA-IL-1 well correlated with the decrease in its receptor binding affinities.     

Neurturin, RET, GFRα-1 and GFRα-2, but not GFRα-3, mRNA are expressed in mice gonads

The gonads are known to produce numerous hormones and also neurotrophins and their receptors. Here we demonstrate expression of glial-cell-line-derived neurotrophic factor (GDNF) family ligands and related receptors in adult mice gonads by in situ hybridization. GDNF mRNA was expressed in the ovary, but was not detectable in testis. Neurturin (NTN), another ligand in this family, gave rise to strong mRNA hybridization signals in a mosaic pattern in the seminiferous tubules of the testis at stages IX-XII and I-II of the cycle. NTN mRNA signals were also found in uterus and the oviduct. In testis, the transducing receptor RET as well as GDNF receptor alpha-1 (GFR)alpha-1 and GFRalpha-2 were distributed in complementary and overlapping patterns, the former at stages XI-XII-I and the latter at stages VII and VIII. GFRalpha-3 could not be detected. Expression of these trophic molecules suggests involvement of GDNF family ligands and related receptor components in reproduction.     

Characterization of three pro-inflammatory cytokines,TNFα1, TNFα2 and IL-1β, in cage-reared Atlantic bluefin tuna Thunnus thynnus

Atlantic bluefin tuna (BFT) (Thunnus thynnus) is of great economic significance for world aquaculture and therefore it is necessary to ensure optimal and sustainable conditions for the farming of this species. Intensive culture of fish may be limited by infectious diseases that can impact on growth performance and cause heavy losses. However, to date there are no reports of cloning and expression analysis of any major immune genes of Atlantic BFT although some immune genes are known in other BFT species. Therefore the aim of this study was to characterize the first cytokine molecules in Atlantic BFT, through: 1) Isolation of full-length cDNA and gene sequences of TNFα1, TNFα2 and IL-1β, 2) comparison of these molecules to known sequences in other vertebrates, especially teleost fish, by multiple sequence alignment, phylogenetic tree analysis and homology modeling; 3) Quantification of in vivo expression of these cytokines in selected tissues in reared BFT over the duration of the farming process. The results indicated that these three cytokines could have value for monitoring Atlantic BFT health status. Curiously, the liver seemed to be an important site of cytokine production during poor health conditions in this species, perhaps reflecting its role as an important organ involved in fish defenses.     

Substrate specificity of protein tyrosine phosphatases 1B, RPTPα, SHP-1, and SHP-2

We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately 2 orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active toward multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY-1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residues on the C-terminal side of pY but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY-1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme's in vivo substrate specificity.     

Development of glycosylated human interleukin-1α, neoglyco IL-1α, coupled with D-galactose monosaccharide: biological activities in vivo

In our previous study, a galactose monosaccharide with C9 spacer was chemically coupled to recombinant human interleukin 1 (rhIL-1) in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. The glycosylated IL-1 exhibited reduced activities in vitro by 10 to 10 000-fold depending upon different aspects of activities addressed. The affinity to type I and II IL-1 receptors were also reduced. In this study we examined a variety of IL-1 activities in vivo, including upregulation of serum levels of IL-6, 1-acid glycoprotein, NOx, corticosterone, downregulation of serum level of glucose, and recovery of peripheral white blood cells (WBCs) from myelosuppression in 5-fluorouracil-treated mice. In contrast to the biological activities in vitro, these activities in vivo were uniformly reduced by only about 10 to 20-fold compared to untreated IL-1.     

Biosynthesis of 1α,2α,3β-trihydroxy-p-menthane by Fusicoccum amygdali

Measurement of isotope ratios in 1α,2α,3β-trihydroxy-p-menthane, which has been biosynthesized in Fusicoccum amygdali from ³H- and ¹⁴C-labelled mevalonate and in its degradation product diosphenol indicates that: (a) four tritium atoms arising from [5-³H₂, 2-¹⁴C]MVA are retained, one more than suggested from the hydroxylation pattern, (b) menth-2-ene-1-ol is generated from an α-terpinyl cation through a 1,3-hydride shift and (c) trans-cleavage of an α-epoxide by hydrolysis gives 1α,2α,3β-trihydroxy-p-menthane.     

Expression of GFRα-1, GFRα-2, and c-Ret mRNAs in rat adrenal gland

Glial cell line-derived neurotrophic factor (GDNF), an important factor for developing and lesioned pre- and postganglionic sympathetic neurons, and its congeners signal through a receptor complex consisting of the tyrosine kinase c-Ret and a lipid-anchored α receptor (GFRα-1-4). Using in situ hybridization we show now that the mRNA for GFRα-2 is abundant in the adult rat adrenal medulla and its chromaffin cells. Coexpression of c-Ret and GFRα-1 mRNA's is restricted to a scarce subpopulation of medullary sympathetic neurons. Both GFRα-1 and GFRα-2 mRNA's are associated with preganglionic nerve trunks in the adrenal cortex. It is conceivable therefore that GDNF and related factors may activate chromaffin and preganglionic Schwann cells through a GFR-α receptor in absence of c-Ret.     

Synthesis and biological activities in vitro and in vivo of glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminyl-galactose

In order to study the effect of glycosylation on its biological activities, and to develop IL-1α with less deleterious effects, recombinant human IL-1α was chemically coupled with N-acetylneuraminic acid (α1-6) galactose (Neu5Ac-Gal). Glycosylated IL-1α (Neu5Ac-Gal-IL-1α) was purified by anion-exchange chromatography and average number of carbohydrate molecules introduced per molecule of IL-1α was 2.5. Neu5Ac-Gal-IL-1α exhibited reduced activities about 1/15-fold compared to IL-1α in all the activities performed in vitro. Binding affinities of Neu5Ac-Gal-IL-1α to Type I and Type II IL-1 receptors were decreased to 1/15 and 1/10, respectively. Neu5Ac-Gal-IL-1α exhibited reduction in activities in vivo, including induction of serum amyloid A and NO$_x$, and down-regulation of serum glucose. However, Neu5Ac-Gal-IL-1α exhibited comparable activity to IL-1α in improvement of the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of Neu5Ac-Gal-IL-1α was relatively high compared to IL-1α. These results indicate that coupling with Neu5Ac-Gal enabled us to develop neoIL-1α with selective activities in vivo. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminic acid exhibits selective activities in vivo and altered tissue distribution

In order to study the effect of glycosylation on its biological activities and to develop IL-1 with less deleterious effects, N-acetylneuraminic acid (NeuAc) with C9 spacer was chemically coupled to human recombinant IL-1. NeuAc-coupled IL-1 (NeuAc-IL-1) exhibited reduced activities in vitro and receptor-binding affinities by about ten times compared to IL-1. In this study, we examined a variety of IL-1 activities in vivo. NeuAc-IL-1 exhibited a marked reduction in the activity to up-regulate serum IL-6, moderate reduction in the activities to up-regulate serum amyloid A and NOx. However, it exhibited comparable activities as IL-1 to down-regulate serum glucose and to improve the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of NeuAc-IL-1 was high compared to IL-1. These results indicate that coupling with NeuAc enabled us to develop neo-IL-1 with selective activities in vivo and enhanced tissue level.     

Bioactivity and pharmacokinetics of two human serum albumin–thymosin α1-fusion proteins, rHSA-Tα1 and rHSA-L-Tα1, expressed in recombinant Pichia pastoris

Thymosin-α1 (Tα1) is indicated for the treatment of certain viral infections, including hepatitis B and C, and cancers, such as melanoma. In this paper, the fusion genes encoding human serum albumin (HSA) and Tα1 with (rHSA-L-Tα1) and without a linker peptide (rHSA-Tα1) were constructed and overexpressed in P. pastoris. Through the process of ion interaction chromatography (Q-Sepharose F.F), hydrophobic interaction chromatography (Phenyl Sepharose HP) and affinity chromatography (Blue Sepharose F.F), the purity of fusion proteins was greater than 97%. In contrast to the reactivity of normal spleen cells to Con A, the data of in vitro murine spleen lymphocytes proliferation experiment suggested that spleen cells achieved a higher degree of T cell maturation after rHSA-L-Tα1, rHSA-Tα1 and Tα1 treatments, respectively. Moreover, rHSA-L-Tα1, rHSA-Tα1 and Tα1 can also antagonize dexamethasone-induced apoptosis of thymocyte sub-populations. In hydrocortisone-induced immunosuppression mice (in vivo experiments), after subcutaneous injections with two fusion proteins and Tα1 for seven consecutive days, the net increment of body weight, the spleen index and the thymus index were significantly improved. Simultaneously, the increase in SOD level and the decrease in MDA level in plasma were observed. The pharmacokinetic data of rHSA-L-Tα1 and rHSA-Tα1 administered in rats showed an improved pharmacokinetic profile with a conspicuous prolonged half life. The analysis of bioactivity and pharmacokinetics suggested that fusion proteins rHSA-L-Tα1 and rHSA-Tα1 were new drug candidates.     

Adipophilin affects the expression of TNF-α, MCP-1, and IL-6 in THP-1 macrophages

Macrophages accumulated in the arterial intima play an important role in the development of atherosclerosis by producing a large number of proinflammatory cytokines which accelerate the disease. Recent studies show that adipophilin might be involved in inflammatory processes in macrophages. In this study, we observe the effect of adipophilin on proinflammatory cytokine expression and secretion in THP-1 macrophages. SiRNA and adipophilin gene overexpression mediated by an pEGFP-C3 vector were used to observe the effect of adipophilin on proinflammatory cytokines in THP-1 macrophages in vitro. Realtime PCR and enzyme-linked immunosorbent assay (ELISA) were applied to detect the production of tumor necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). It was found that acetylated low-density lipoprotein (AcLDL), pioglitazone [a peroxisome proliferator-activated receptor γ (PPARγ) agonist] increased adipophilin expression in macrophages, while glucose had no such affect. It was also shown that adipophilin augments TNF-α, MCP-1, and IL-6 expression in AcLDL induced macrophages. Our results suggest that adipophilin augment inflammation in macrophages, which might be one role of adipophilin in atherosclerosis.     

α_1,α_2受体的突触前后定位和亚亚型划分

突触前和突触后都有α_1和α_2受体。对突触前α_1受体的功能有不同看法,可能与突触前α_2受体类似;突触后α_2受体的功能亦与突触后α_1受体类似,但有差别。α_1受体可再划分出高亲和力α_1受体与低亲和力α_1受体:α_(1a)、α_(1b)受体;还有人提出α_(1(?))受体,依赖和不依赖细胞外钙的α_1受体的概念。α_2受体可再划分为低亲和力α_2受体(α_(2L))和高亲和力α_2受体(α_(2H))。