Identification of the tree shrew ATP-binding cassette transporter A1 (ABCA1) and its expression in tissues : cDNA sequence and expression of tree shrew ABCA1.

ATP-binding cassette transporter A1 (ABCA1) modulates plasma levels of high density lipoprotein (HDL), a cardiovascular protecting factor. Tree shrew was considered to be an animal protected from atherosclerosis characterized by high proportion of HDL in plasma. The cDNA clones and expression of tree shrew ABCA1 was identified using SMART-RACE and Real-Time PCR techniques respectively. The nucleotide sequence of tree shrew ABCA1 covered 7,762 bp, including a 6,786 bp coding region which encoded a 2,261 amino acids protein with the high identity to human ABCA1 (95%). Tree shrew ABCA1 was expressed in various tissues, the highest in lung, followed by liver, kidney, spleen and cardiac muscle in turn from high to medium expression levels. This pattern was partially different from that of human ABCA1 which was low in kidney and cardiac muscle. This work could shed new light on its role of ABCA1 in the distinctive HDL metabolism in tree shrew.

[cDNA cloning and prokaryotic expression of human cholesteryl ester transfer protein and preparation of its antiserum].

AIM: To clone human cholesteryl ester transfer protein (CETP) cDNA, express and purify human CETP in E.coli and prepare CETP-specific rabbit antiserum. METHODS: by RT-PCR method, the encoding sequence of human cholesteryl ester transfer protein (CETP) was cloned into pET-30b(+) vector. Then BL21 (DE3) of E.coli transformed with recombinant vector pET-CETP was induced to express CETP in high level by IPTG. The expressed protein was purified from SDS-polyacrylamide gel, and the antiserum against CETP was raised in rabbit. The titer and specificity of rabbit antiserum were evaluated by ELISA, Western blot and immunofluorescence assay. RESULTS: The results of SDS-PAGE showed that CETP was expressed in the form of inclusion bodies in BL21(DE3) and the best expression time was about 4 hours. The titer of the rabbit antiserum prepared with CETP purified from SDS-PAGE was 1:5. 12 x 10(5) and the antiserum reacted specifically with CETP expressed in BL21 (DE3) and COS7 cells. CONCLUSION: The preparation of the specific rabbit antiserum against CETP will be valuable for the study on the structure and function of human CETP.

Haplotype analyses of the APOA5 gene in patients with familial combined hyperlipidemia.

BACKGROUND: Familial combined hyperlipidemia (FCH) is the most common genetic lipid disorder with an undefined genetic etiology. Apolipoprotein A5 gene (APOA5) variants were previously shown to contribute to FCH. The aim of the present study was to evaluate the association of APOA5 variants with FCH and its related phenotypes in Dutch FCH patients. Furthermore, the effects of variants in the APOA5 gene on carotid intima-media thickness (IMT) and cardiovascular disease (CVD) were examined. MATERIALS AND METHODS: The study population consisted of 36 Dutch families, including 157 FCH patients. Two polymorphisms in the APOA5 gene (-1131T>C and S19W) were genotyped. RESULTS: Haplotype analysis of APOA5 showed an association with FCH (p=0.029), total cholesterol (p=0.031), triglycerides (p<0.001), apolipoprotein B (p=0.011), HDL-cholesterol (p=0.013), small dense LDL (p=0.010) and remnant-like particle cholesterol (p=0.001). Compared to S19 homozygotes, 19W carriers had an increased risk of FCH (OR=1.6 [1.0-2.6]; p=0.026) and a more atherogenic lipid profile, reflected by higher triglyceride (+22%) and apolipoprotein B levels (+5%), decreased HDL-cholesterol levels (-7%) and an increased prevalence of small dense LDL (16% vs. 26%). In carriers of the -1131C allele, small dense LDL was more prevalent than in -1131T homozygotes (29% vs. 16%). No association of the APOA5 gene with IMT and CVD was evident. CONCLUSION: In Dutch FCH families, variants in the APOA5 gene are associated with FCH and an atherogenic lipid profile.

[Structure analysis of apolipoprotein B 3' variable number of tandem repeats].

OBJECTIVE: To study the distribution, frequency and structure of variable number of tandem repeats (VNTR) in 3' region of apoB gene in Chinese population. METHODS: Genomic DNA was extracted from peripheral blood obtained under consent from randomly-chosen 522 individuals who came to the hospital for physical examination, and used to screen for polymorphisms of 3' VNTR of the apoB gene by employing polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), gradient polyacrylamide gel electrophoresis, cloning and sequencing. RESULTS: Sixteen types of alleles of apoB 3oVNTR were identified, among which heterozygotes were more than homozygotes. The biggest allele is HVE58, and the smallest one is HVE22. HVE34 had the highest frequency (40.4%), followed by HVE32 (34.7%). This showed significant difference from the allelic distribution of other populations (Caucasian and Swedish). Through sequencing of 60 alleles, a new isomer (Y-A=ATAATTAAATATTT) and four new types of alleles were found. CONCLUSION: The Chinese population we studied had a higher frequency of small alleles and showed a difference in allelic structure and frequency distribution from European and American in this populations.

[Study on the relationship between smoking, alcohol intake and hyperlipidemia in fishermen].

OBJECTIVE: To identify the relationship between smoking, alcohol intake and hyperlipidemia in fishermen. METHODS: 115 fishermen were randomly recruited and divided into case and control groups according to the result of blood lipoprotein. A questionnaire was used to record general information and the history of smoking and alcohol intake. Statistics were gathered to compare the difference of lipoprotein and apolipoprotein level between exposure and control groups and to calculate the OR value of smoking and alcohol intake. RESULTS: The OR of smoking was 3.417 (95% CI: 1.132 - 10.308), with significant dosage-effect relationship between smoking index and hyperlipidemia. The serum low density lipoprotein-cholesterol (LDL-C) and apolipoprotein (apo) B levels in smoking group was higher than that of control group. The OR value of alcohol intake at early age (early than 20) were 3.275 (95% CI: 1.249 - 8.580) and 4.016 (95% CI: 1.475 - 10.952) respectively. The LDL-C, apoB, the serum total cholesterol (TC)/high density lipoprotein-cholesterol (HDL-C) levels in alcohol abuse group were higher than that of control group. CONCLUSION: Smoking and alcohol abuse were important risk factors of hyperlipidemia, through changing the level of LDL-C and apoB. There was synergistic action between smoking and alcohol abuse in the development of hyperlipidemia.

[Analysis of lecithin-cholesterol acyltransferase cDNA and protein sequence from tree shrew].

OBJECTIVE: To acquire cDNA sequence of lecithin-cholesterol acyltransferase (LCAT) from tree shrew and analyze the sequence structure. METHODS: The first strand cDNA was acquired by reverse transcription using mRNA from tree shrew liver as template. By the method of SMART RACE PCR, tree shrew LCAT cDNA was acquired and deduced its amino acids sequence. The sequence and structure of tree shrew LCAT cDNA and amino acid were analyzed and predicted by the molecular software. RESULTS: Tree shrew LCAT cDNA is composed of 1,340 bp, including 2 bp 5' untranslated region (5' UTR), 1,320 bp open reading frame (ORF) which encodes protein precursor of 440 amino acids (24 amino acids signal peptide and 416 amino acids mature peptide), and 18 bp 3' untranslated region (3'UTR). The stop codon is TAA and there is a poly (A) signal sequence AATAAA and a 25 bp poly (A) tail. Tree shrew LCAT cDNA sequence has been accepted by GenBank as a new gene, accession number AF272861 and its homology with human and baboon was 90% and 89%, respectively. CONCLUSION: The sequence of LCAT cDNA in tree shrew has high identity with that of human and other experimental animal species.

The cDNA Sequence and Its Tissue Expression of Tree Shrew Apolipoprotein CI.

The mRNA was isolated and purified from tree shrew (TS) liver tissue. A cDNA library of the liver tissue was then constructed by using the mRNA as the template by reverse transcription. Two apolipoprotein CI (apoCI) cDNA clones were identified in the library with an anti-serum to TS apoCI. Sequencing and analysizing of the clones showed that both were the apoCI cDNA sequences in which the larger one was determined as 380 nucleotides. It contains 21 bp and 95 bp in 5' and 3' untranslated regions respectively, and 264 bp in an open reading frame, encoding an 88 aa apoCI precursor (a 26 aa signal peptide, and a 62 aa mature protein whose length is the same as those of rat, mouse and dog, but longer than those of human and baboon by 5 aa residues). The function domains in the protein sequence deduced from the cDNA were predicted by comparison of conservative regions in apoCIs from different species. The results of Northern blot indicated in TS the presence of apoCI mRNA not just mainly in the liver but also found in the intestine, suggesting the mRNA distribution different from those in other mammals and primates.