Does the AD7c-NTP locus encode a protein?

AD7c-NTP, the only known protein entirely encoded by tandem and nested cassettes of Alu repetitive elements, is reportedly over-expressed in brains of Alzheimer's disease patients [de la Monte et al., J. Clin. Invest. 15 (1997)]. Based on these findings a commercial diagnostic assay ("7c Gold"/"AlzheimAlert" ) has been developed. We analyzed the published cDNA sequence and compared it to corresponding EST clones as well as the genomic sequences of human and chimpanzee. We come to the conclusion that the existence of the gene and in particular the predicted protein is inconsistent with EST and genomic data. Previously published data need to be reassessed.     

Molecular cloning and characterization of a novel human kinase gene,PDIK1L

We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named ’PDLIM1 interacting kinase 1 like’ (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser locatedPDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.     

Molecular cloning of human growth inhibitory factor cDNA and its down-regulation in Alzheimer''s disease.

In previous studies, we discovered a growth inhibitory factor (GIF) that was abundant in normal human brain, but greatly reduced in Alzheimer's disease (AD) brain. Molecular cloning of a full-length cDNA for human GIF revealed that the GIF had striking homology to metallothioneins. Furthermore, it was determined that the GIF gene was on chromosome 16, as are the metallothionein genes. GIF, in contrast to metallothioneins, was found to be expressed exclusively in the nervous system. The GIF protein produced by Escherichia coli harboring the GIF cDNA in a prokaryotic expression vector inhibited the growth of neonatal rat cortical neurons. These results indicate that GIF is a new member of the metallothionein family with distinct tissue-specific expression and functions. Northern blot analysis revealed that expression of the GIF mRNA is drastically decreased in AD brains. The result raises the possibility that down-regulation of the GIF gene in AD brain plays an important role in the pathogenesis of AD.     

Combining serum and urine biomarkers in the early diagnosis of mild cognitive impairment that evolves into Alzheimer’s disease in patients with the apolipoprotein E ε4 genotype

Abstract

Possession of the apolipoprotein E (APOE) ε4 genotype is a major predictor of progression to Alzheimer’s disease (AD), particularly in patients with mild cognitive impairment (MCI). However, the use of APOE genotyping in the diagnosis of MCI is limited due to its low sensitivity and specificity, which often results in a high false-positive rate. In this study, we found that there was a significant decrease in serum BDNF and notable increase in urine AD7c-NTP in MCI patients who harbored the APOE ε4 allele. Both serum BDNF and urine AD7c-NTP had higher positive predictive values and were more sensitive biomarkers of MCI. Additionally, a testing strategy employing serum BDNF and urine AD7c-NTP revealed increases in sensitivity, positive and negative predictive values, and predictive ability compared with the use of either biomarker alone, suggested that combinatorial detection might have great potential for translation to the clinic.     

Identification and characterization of a novel Cut family cDNA that encodes human copper transporter protein CutC

Copper is an essential heavy metal trace element that plays important roles in cell physiology. The Cut family was associated with the copper homeostasis and involved in several important metabolisms, such as uptake, storage, delivery, and efflux of copper. In this study, a novel Cut family cDNA was isolated from the human fetal brain library, which encodes a 273 amino acid protein with a molecular mass of about 29.3 kDa and a calculated pI of 8.17. It was named hCutC (human copper transporter protein CutC). The ORF of hCutC gene was cloned into pQE30 vector and expressed in Escherichia coli M15. The secreted hCutC protein was purified to a homogenicity of 95% by using the Ni-NTA affinity chromatography. RT-PCR analysis showed that the hCutC gene expressed extensively in human tissues. Subcellular location analysis of hCutC-EGFP fusion protein revealed that hCutC was distributed to cytoplasm of COS-7 cells, and both cytoplasm and nucleus of AD293 cells. The results suggest that hCutC may be one shuttle protein and play important roles in intracellular copper trafficking.     

Differential display reveals downregulation of the phospholipid transfer protein (PLTP) at the mRNA level in brains of patients with Down syndrome

The phospholipid transfer protein (PLTP) shows a wide variety of functions including transfer of phospholipids and other lipid-like substances. Performing gene hunting in brain of patients with Down syndrome (DS) we detected the absence of a fragment identified as PLTP. Cerebellum of 4 controls, 7 patients with DS, 5 patients with Alzheimer's disease (AD) were used for differential display and for quantification of mRNA steady state levels of the isomer PLTP-1 by blotting methods. Differential display showed the absence of a cDNA fragment and cloning, sequencing and gene bank work revealed 100% homology with human PAC 337018 on chromosome 20q containing the PLTP gene. The PLTP gene in turn consists of at least three different PLTP-isomers. Based on these results, a 450 bp cDNA fragment of the PLTP-isomer I (PLTP I) was isolated and amplified by PCR, serving as probe for the PLTP-1 isomer and its expression level was found to be significantly reduced in cerebellum of patients with DS. Biologically, the downregulation of PLTP maybe involved in the pathology of DS as phospholipids not only are of importance for membrane biogenesis and structure but also in the regulation of cellular metabolism, signaling and growth. In the brain, phospholipids in addition are integral constituents of myelins and synaptosomes (Johnson etc) and deficient PLTP levels may account for the deteriorated functions described to occur in DS brain.     

Cloning and expression of sterol Delta 14-reductase from bovine liver.

Biosynthesis of cholesterol represents one of the fundamental cellular metabolic processes. Sterol Delta 14-reductase (Delta 14-SR) is a microsomal enzyme involved in the conversion of lanosterol to cholesterol in mammals. Amino-acid sequence analysis of a 38-kDa protein purified from bovine liver in our laboratory revealed > 90% similarity with a human sterol reductase, SR-1, encoded by the TM7SF2 gene, and with the C-terminal domain of human lamin B receptor. A cDNA encoding the 38-kDa protein, similar to human TM7SF2, was identified by analysis of a bovine expressed sequence tag (EST) database. The cDNA was synthesized by RT-PCR, cloned, and sequenced. The cDNA encodes a 418 amino-acid polypeptide with nine predicted transmembrane domains. The deduced amino-acid sequence exhibits high similarity with Delta 14-SR from yeasts, fungi, and plants (55-59%), suggesting that the bovine cDNA encodes Delta 14-SR. Northern blot analysis of bovine tissues showed high expression of mRNA in liver and brain. The polypeptide encoded by the cloned cDNA was expressed in COS-7 cells. Immunofluorescence analysis of transfected cells revealed a distribution of the protein throughout the ER. COS-7 cells expressing the protein exhibited Delta 14-SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes Delta 14-SR and provide evidence that the human TM7SF2 gene encodes Delta 14-SR.     

人类新基因ACBP5cDNA的克隆及其同源物在小鼠/鸡胚发育过程中的表达

利用电子克隆的方法寻找具有重要结构域的人类新基因ACBP5 ,根据得到的序列信息用RT PCR的方法获得全长基因 .通过生物信息学方法预测其结构 ,采用整体原位杂交和组织RT PCR的实验方法 ,在小鼠和鸡胚胎实验模型中研究该基因在发育过程中的表达情况 ,并对其功能进行初步的预测 ,获得一个含有乙酰辅酶A结合蛋白 (acyl CoAbindingprotein ,ACBP)结构域的人类新基因ACBP5 .ACBP5基因的cDNA长度为 10 83bp ,生物信息学方法预测其定位在人第 1号染色体上 ,包含 7个外显子 ,6个内含子 ,包含一个 35 4bp的完整阅读框架 ,编码一个 118个氨基酸残基的蛋白 .在以小鼠胚胎和鸡胚为模型的整体原位杂交中 ,以ACBP5基因全长编码区为探针的结果均显示该基因在胚胎头部特异表达 ,并且主要集中在中脑与间脑之间的峡部 .成体小鼠的组织RT PCR的结果显示 ,ACBP5的同源基因在各组织中均有表达 .这提示ACBP5基因在不同物种中的表达可能比较保守 ,并与头部发育有密切关系 ,同时也对维持细胞的正常功能起到重要的作用 .     

Isolation of a ubiquitin-like (UBL5) gene from a screen identifying highly expressed and conserved iris genes

We have screened a human adult iris cDNA library to identify genes that are highly expressed and conserved between humans and pigs. We identified human iris cDNAs that hybridized at high stringency to a porcine choroidal ring cDNA probe. Of 1568 human iris cDNAs examined, 176 were found to have high expression in porcine choroidal rings. One of the 176 clones was identified as a previously uncharacterized cDNA that we have named the Ubiquitin-like 5 gene (UBL5). The UBL5 gene is located on chromosome 19p13.2, and its genomic structure has been examined. There is a UBL5 pseudogene on chromosome 17p11.2. We have also found homologues to the UBL5 gene in Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Northern blot analysis of the Ubiquitin-like gene 5 revealed expression in every tissue tested, with the highest levels of RNA expression in heart, skeletal muscle, kidney, liver, iris, and lymphoblasts. Intracellular localization experiments in COS-7 cells showed that the recombinant UBL5 protein is cytoplasmic. Western analysis demonstrated that the recombinant UBL5 protein is approximately 9 kDa, as predicted from the cDNA. A comparison between UBL5 and its homologues with other Ubiquitin-like proteins and Ubiquitin, using the PROTDIST program, suggests that the UBL5 genes are a separate class of Ubiquitin-like genes. Further characterization of the UBL5 gene will determine the function of the encoded protein and whether it is a candidate for ocular disease.     

Up-regulation of calcineurin Abeta mRNA in the Alzheimer's disease brain: assessment by cDNA microarray

Recent advances in cDNA microarray technology have made it possible to analyze expression of more than 8000 genes. Using this technology, gene expression in the hippocampus containing neurofibrillary tangle-associated lesions from an Alzheimer's disease (AD) patient was compared with expression in the parietal cortex from the same patient that lacked these lesions. We also compared gene expression using a control brain. The top 20 named genes significantly up-regulated or down-regulated only in the AD brain were determined. The most up-regulated gene proved to be calcineurin Abeta mRNA (CAbeta). In situ hybridization histochemistry revealed that CAbeta was significantly up-regulated in pyramidal neurons of the hippocampus in the AD brain. RT-PCR analysis revealed that CAbeta was up-regulated in the hippocampus from two out of three AD brains while there were no changes in three control brains. Our study suggests that CAbeta may play a crucial role in the pathophysiological mechanisms in AD.     

A novel splice variant of human gene NPL, mainly expressed in human liver, kidney and peripheral blood leukocyte.

From the human fetal brain cDNA library constructed by our lab, a novel variant cDNA of a human gene was successfully cloned and identified. Because the gene has been named N-acetylneuraminate pyruvate lyase (NPL), accordingly we term our splice variant NPL_v2. The cDNA of NPL_v2 has a full-length open reading frame (ORF) from the nucleotide position 320 to 1225 that encodes a protein comprising 301 amino acids. SMART analysis showed that our hypothetical protein has one dihydrodipicolinate synthase (DHDPS) domain. Phosphorylation analysis of the deduced protein show that there are five phosphorylation sites including three "serine" and two "threonine" at the region that are not found in other splice variant. RT-PCR experiment revealed that our splice variant of the gene is mainly expressed in human placenta, liver, kidney, pancreas, spleen, thymus, ovary, small intestine and peripheral blood leukocyte.     

Alzheimer''s disease amyloidogenic glycoprotein: expression pattern in rat brain suggests a role in cell contact.

The cloned cDNA encoding the rat cognate of the human A4 amyloid precursor protein was isolated from a rat brain library. The predicted primary structure of the 695-amino acid-long protein displays 97% identity to its human homologue shown previously to resemble an integral membrane protein. The protein was detected in rodent brain and muscle by Western blot analysis. Using in situ hybridization and immunocytochemistry on rat brain sections, we discovered that rat amyloidogenic glycoprotein (rAG) and its mRNA are ubiquitously and abundantly expressed in neurons indicating a neuronal original for the amyloid deposits observed in humans with Alzheimer's disease (AD). The protein appears in patches on or near the plasma membranes of neurons suggesting a role for this protein in cell contact. Highest expression was seen in rat brain regions where amyloid is deposited in AD but also in areas which do not contain deposits in AD. Since amyloid deposits are rarely observed in rat brain, we conclude that high expression of AG is not the sole cause of amyloidosis.     

Cloning,expression and genomic structure of a novel human GNB2L1 gene,which encodes a receptor of activated protein kinase C (RACK)*

During a large-scale screen of a human fetal brain cDNA library, a novel human gene GNB2L1 encoding a novel RACK (receptor of activated protein kinase C) protein was isolated and sequenced. The cDNA is 1142 bp long and has a predicted open reading frame encoding 316 aa. The predicted protein shows higher similarity to rat RACK1 and many RACK proteins of different organisms including Drosophila, C. elegans, mouse, rat, human, C. fasciculata, zebrafish, A. thaliana, S. cerevisiae and so on, suggesting it is conserved during evolution. The gene was mapped to human chromosome 5q35.3, the telomer position of chromosome 5q, in which the disease gene for early-onset primary congenital lymphedema was mapped. Also, 5q35.3 is a frequently reported location for cytogenetic and molecular abnormalities in renal cell carcinomas. The gene has 8 exons and 7 introns. It is expressed ubiquitously in many human tissues detected by northern blot analysis and RT-PCR.     

Increased expression of the lysosomal cholesterol transporter NPC1 in Alzheimer's disease

The Niemann-Pick type C1 (NPC1) protein mediates the trafficking of cholesterol from lysosomes to other organelles. Mutations in the NPC1 gene lead to the retention of cholesterol and other lipids in the lysosomal compartment, and such defects are the basis of NPC disease. Several parallels exist between NPC disease and Alzheimer's disease (AD), including altered cholesterol homeostasis, changes in the lysosomal system, neurofibrillary tangles, and increased amyloid-beta generation. How the expression of NPC1 in the human brain is affected in AD has not been investigated so far. In the present study, we measured NPC1 mRNA and protein expression in three distinct regions of the human brain, and we revealed that NPC1 expression is upregulated at both mRNA and protein levels in the hippocampus and frontal cortex of AD patients compared to control individuals. In the cerebellum, a brain region that is relatively spared in AD, no difference in NPC1 expression was detected. Similarly, murine NPC1 mRNA levels were increased in the hippocampus of 12-month-old transgenic mice expressing a familial AD form of human amyloid-beta precursor protein (APP) and presenilin-1 (APP/PS1tg) compared to 12-month-old wild type mice, whereas no change in NPC1 was detected in mouse cerebellum. Immunohistochemical analysis of human hippocampus indicated that NPC1 expression was strongest in neurons. However, in vitro studies revealed that NPC1 expression was not induced by transfecting SK-N-SH neurons with human APP or by treating them with oligomeric amyloid-beta peptide. Total cholesterol levels were reduced in hippocampus from AD patients compared to control individuals, and it is therefore possible that the increased expression of NPC1 is linked to perturbed cholesterol homeostasis in AD.