An alternatively spliced soluble TNF-alpha receptor is associated with metabolic disorders: a replication study

In a previous study, we identified a biologically active form of tumor necrosis factor-alpha receptor 2 (sTNFR2) produced by differential splicing (DS-TNFR2) which antagonized TNF-alpha biological activity. Obesity, insulin resistance and type 2 diabetes are linked to increased TNF-alpha action. We hypothesized that subjects with detectable DS-TNFR2 would be protected from developing obesity and related metabolic disorders. Thus, we investigated if circulating DS-TNFR2 concentration was associated with components of the so-called metabolic syndrome among 269 consecutive subjects from the general population. DS-TNFR2 was measured using a monoclonal antibody against an epitope present in TNFR2 (first 14 residues of the juxtamembrane region) but predicted to be absent in soluble proteolytic cleavage-produced TNFR2. Plasma DS-TNFR2 concentration was significantly decreased among patients with glucose intolerance or type 2 diabetes mellitus (p=0.026). DS-TNFR2 tended to be associated with fasting and post-load glucose (both r=-0.11, p=0.054), and with diastolic blood pressure in men (r=-0.16, p=0.07). Serum DS-TNFR2 concentration was significantly associated with LDL cholesterol (r=-0.28, p=0.002), uric acid (r=-0.13, p=0.04) and with blood glycated hemoglobin (r=-0.13, p=0.04). DS-TNFR2 declined with increased number of components of the metabolic syndrome (p=0.03). Those subjects with 2 or more components had significantly decreased circulating DS-TNFR2 levels (0.96+/-2.2 versus 1.7+/-3.2, p=0.033). In summary, the circulating concentration of DS-TNFR2 seems to be inversely linked to metabolic disorders, hinting at a possible anti-inflammatory role.     

The growth hormone-binding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor

A cDNA clone isolated from rat liver was demonstrated to encode a soluble, secreted growth hormone (GH)-binding protein consistent with the properties of the newly discovered serum GH-binding protein. The protein coding region of this cDNA was identical in sequence to the extracellular domain of the rat liver GH receptor up to three amino acids before the putative transmembrane domain. At this point, an additional 17 amino acids were encoded in the GH-binding protein before a stop codon was encountered. This cDNA clone was shown to be representative of the structure of the mRNA present in rat liver. These results suggest that the mechanism for production of the rat serum GH-binding protein is by alternative splicing of the gene for the rat GH receptor.     

A novel species-specific RNA related to alternatively spliced amyloid precursor protein mRNAs.

Using an S1 nuclease protection assay, we have identified a novel "variant" Amyloid Precursor Protein (APP) RNA in human brain which is 3-6-fold more abundant than APP-770, but less abundant than APP-751 or APP-695. This variant, referred to as amyloid precursor-related protein 365 (APRP-365), is not detected in mouse and rat brain RNAs. A 1.6 kilo-basepair cDNA clone corresponding to this variant APP RNA predicts the existence of a 365 amino acid protein that is similar to the amino-terminal end of APP-770 but lacks the beta-amyloid peptide and any hydrophobic transmembrane spanning domains. In a modified polymerase chain reaction (PCR), we used amplification of reverse transcribed mRNA to confirm and extend our S1 observations. Together, the features of APRP-365 suggest that the human variant is a soluble protein containing a Kunitz protease inhibitor domain.     

Identification of an alternatively spliced mRNA transcript of human betacellulin lacking the C-loop of the EGF motif and the transmembrane domain

This paper describes the cloning and characterization of a novel cDNA encoding a short form of betacellulin (BTC-beta), and reports the expression of this mRNA in a variety of human tissues and cell types. BTC-beta is likely the result of alternative splicing. This splicing event leads to the generation of an mRNA encoding an unusual BTC precursor in which the C-loop of the EGF domain and the transmembrane domain are deleted while the remainder of the mature molecule is fused in-frame to the C-terminal cytoplasmic tail.     

前列腺特异性膜抗原新型剪接变异体PSME基因及蛋白的生物信息学分析

目的通过对PSME的生物信息学分析,更多的了解该基因及蛋白结构与功能的相关信息。方法运用生物信息学方法分析PSME基因结构,序列及其编码蛋白的理化性质和结构与功能特征,蛋白相互作用网络以及抗原表位。结果用ExPASy的Computer PI/Mw、SMART等软件对其氨基酸序列进行分析,该蛋白的PI值为6.38,相对分子质量约为78 000。二级结构中α螺旋(H)占34.66%,β折叠(E)占12.93%,无规卷曲占52.41%。PSME信号肽位于1～22位氨基酸,150～248位为肽酶结构域,639～703位为转铁蛋白受体二聚体,且含有多个糖基化位点。通过DNA Star软件分析得到了PSME蛋白的抗原表位。结论利用生物信息学预测出的结构和功能信息,能为PSME蛋白的相关研究提供信息基础。     

CADM1 is expressed as multiple alternatively spliced functional and dysfunctional isoforms in human mast cells

Cell adhesion molecule 1 (CADM1) is implicated in the pathogenesis of several diseases and is responsible for adhesion and survival of mast cells (MCs). Differential expression of CADM1 isoforms was found in different species. We previously cloned SP4, SP1, SP6 and a dysfunctional isoform from human lung MCs (HLMCs) and the MC line HMC-1. The aim of this study was to identify all isoforms expressed in human MCs. The functional isoforms SP4, SP1, SP6 and SP3, with alternative splicing between exons 7/11, were detected in human MCs by RT-PCR. Two dysfunctional isoforms with alternative splicing of cryptic exons A and B between exons 1/2, leading to premature termination of translation, were found in ～40% of MC specimens. Sequencing of genomic DNA showed that splicing of cryptic exon B did not result from specific SNPs within this exon or its putative splice branch point. Highly glycosylated CADM1 (～105 kDa) was detected by western blotting, but an extracellular domain (～95 kDa) was found only in the culture medium from HLMCs, but not HMC-1 cells, indicating differential protein expression. Transfection of SP1 and SP6, but not SP4, reduced adhesion of HMC-1 cells to human lung fibroblasts but not airway smooth muscle cells. Hence, dysfunctional and functional CADM1 isoforms are found in human MCs. The longer SP1 and SP6 were most evident in differentiated HLMCs and displayed differential adhesion compared to SP4. These multiple isoforms are likely to contribute to MC function in both health and disease.     

The genomic structure and a novel alternatively spliced form of porcine pTalpha chain

A complete genomic nucleotide sequence for porcine pTalpha gene was obtained from a BAC clone, which revealed a novel exon 2 missing in human and murine counterparts. Cattle and dog genomic sequences showed the counterparts corresponding to porcine exon 2. Using thymocyte RNA and RT-PCR, three types of porcine pTalpha-chain cDNA sequences, pTalpha1, pTalpha2 and pTalpha3, were obtained. These three different cDNA sequences were alternatively spliced products with pTalpha1 consisting of exons 1, 2, 3, 4, and 5, pTalpha2 consisting of exons 1, 2, 4, and 5, and pTalpha3 consisting of exons 1, 2, 3 and the intron down stream of exon 3. pTalpha1 and pTalpha2 correspond to previously reported pTalphaa, and pTalphab, respectively, and pTalpha3 is reported for the first time. Using RT-PCR, pTalpha3 appeared expressed predominantly in the thymocyte RNA. The chromosome location of pTalpha was investigated using Radiation Hybrid Map and FISH, both of which revealed the location at SSC7q11-q12.     

Identification and characterization of a functional, alternatively spliced Toll-like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens

Based upon the recognition of antiviral compounds and single stranded viral RNA the Toll-like receptors TLR7 and TLR8 are suggested to play a significant role in initiating antiviral immune responses. Here we report the molecular characterization of the chicken TLR7/8 loci which revealed an intact TLR7 gene and fragments of a TLR8-like gene with a 6-kilobase insertion containing chicken repeat 1 (CR1) retroviral-like insertion elements. The chicken TLR7 gene encodes a 1047-amino-acid protein with 62% identity to human TLR7 and a conserved pattern of predicted leucine-rich repeats. Highest levels of chicken TLR7 mRNA were detected in immune-related tissues and cells, especially the spleen, caecal, tonsil and splenic B cells. Alternative spliced forms of TLR7 mRNA were identified in chicken, mouse and human and expressed in similar tissues and cell types to the major form of chicken TLR7. The chicken TLR7+ HD11 cell line and fresh splenocytes produced elevated levels of interleukin-1beta (IL-1beta) mRNA after exposure to the agonists R848 and loxoribine. Interestingly, none of the TLR7 agonists stimulated increased type I interferon (IFN) mRNA whereas poly(I:C) (a TLR3 agonist) up-regulated both chicken IFN-alpha and chicken IFN-beta mRNA. In contrast, TLR7 agonists, particularly R848 and poly(U) stimulated up-regulation of chicken IL-1beta, and chicken IL-8 mRNAs more effectively than poly(I:C). Stimulation of chicken TLR7 with R848 was chloroquine sensitive, suggesting signalling within an endosomal compartment, as for mammalian TLR7. The deletion of TLR8 in galliforms, accompanied with the differential response after exposure to TLR7 agonists, offers insight into the evolution of vertebrate TLR function.     

新型前列腺特异膜抗原剪接变异体的发现及临床意义初步探讨

目的:探讨前列腺特异膜抗原（PSMA）及其与前列腺癌发生、发展的关系，寻求更为特异的前列腺癌诊断和治疗的靶点。 方法:采用RT-PCR和DNA测序技术，克隆PSMA基因新的剪接变异体，并根据其序列信息，设计特异性引物，检测其在不同病变前列腺组织及不同组织来源肿瘤细胞中的表达。 结果:发现了一种新的PSMA剪接变异体，其在前列腺癌、前列腺增生及正常前列腺组织中的表达率分别为92.6％、78.8％及10.0％，且特异表达于前列腺癌LNCaP细胞株，而在前列腺癌PC3细胞株、膀胱癌、肾癌、肝癌细胞株中均不表达。 结论:发现了一种新型PSMA剪接变异体（定名为PSMA5），并证实该变异体与前列腺癌及前列腺增生有明显相关性，为研究前列腺癌的发生机制和寻求前列腺癌特异性诊治靶点提供了新的线索。