Characteristic, polymorphism and expression distribution of LCAT gene in a Mongolian gerbil model for hyperlipidemia.

This study aims to evaluate the genetic basis and activity of lecithin cholesterol acyltransferase (LCAT) in a novel Mongolian gerbil model for hyperlipidemia. Gerbils may be susceptible to high fat and cholesterol (HF/HC) diets, which can rapidly lead to the development of hyperlipidemia. Approximately 10-30% of gerbils that are over 8months old and fed controlled diets spontaneously develop hyperlipidemia. Using the HF/HC diet model, we detected triglycerides (TG), total cholesterol (TC), HDL (high density lipoprotein)-C, LDL (low density lipoprotein)-C and LCAT in both old (>8months) and young gerbils. The TC and HDL-C levels were two times higher in old gerbils compared with young gerbils (P<0.01). However, in the old group the LCAT activity fell slightly compared with the normal lipidemia group. It is reasonable to hypothesize that this may be associated with single nucleotide polymorphisms of the LCAT gene. We cloned this gene to investigate the sensitivity of the gerbil to the HF/HC diet and spontaneous hyperlipidemia. The entire LCAT gene was cloned by splicing sequences of RACE (rapid amplification of cDNA ends) and nest-PCR products (AN: KC533867.1). The results showed that the 3683base pair gene consists of six exons and five introns. The LCAT protein consists of 444 amino acid (AA) residues, which are analogous to the human LCAT gene, and includes 24 signal peptide AA and 420 mature protein AA. Expression of LCAT was detected in the kidney, spleen and adrenal tissue, apart from the liver, by immunohistochemistry. The abundance of the protein was greater in the older group compared with the control group. Polymorphisms were analyzed by PCR-SSCP (PCR-single-strand conformation polymorphism) but none were found in 444 animals of the ZCLA closed population (a Chinese cultured laboratory gerbil population).

Reduced 64Cu uptake and tumor growth inhibition by knockdown of human copper transporter 1 in xenograft mouse model of prostate cancer.

UNLABELLED: Copper is an element required for cell proliferation and angiogenesis. Human prostate cancer xenografts with increased (64)Cu radioactivity were visualized previously by PET using (64)CuCl2 as a radiotracer ((64)CuCl2 PET). This study aimed to determine whether the increased tumor (64)Cu radioactivity was due to increased cellular uptake of (64)Cu mediated by human copper transporter 1 (hCtr1) or simply due to nonspecific binding of ionic (64)CuCl2 to tumor tissue. In addition, the functional role of hCtr1 in proliferation of prostate cancer cells and tumor growth was also assessed. METHODS: A lentiviral vector encoding short-hairpin RNA specific for hCtr1 (Lenti-hCtr1-shRNA) was constructed for RNA interference-mediated knockdown of hCtr1 expression in prostate cancer cells. The degree of hCtr1 knockdown was determined by Western blot, and the effect of hCtr1 knockdown on copper uptake and proliferation were examined in vitro by cellular (64)Cu uptake and cell proliferation assays. The effects of hCtr1 knockdown on tumor uptake of (64)Cu were determined by PET quantification and tissue radioactivity assay. The effects of hCtr1 knockdown on tumor growth were assessed by PET/CT and tumor size measurement with a caliper. RESULTS: RNA interference-mediated knockdown of hCtr1 was associated with the reduced cellular uptake of (64)Cu and the suppression of prostate cancer cell proliferation in vitro. At 24 h after intravenous injection of the tracer (64)CuCl2, the (64)Cu uptake by the tumors with knockdown of hCtr1 (4.02 ± 0.31 percentage injected dose per gram [%ID/g] in Lenti-hCtr1-shRNA-PC-3 and 2.30 ± 0.59 %ID/g in Lenti-hCtr1-shRNA-DU-145) was significantly lower than the (64)Cu uptake by the control tumors without knockdown of hCtr1 (7.21 ± 1.48 %ID/g in Lenti-SCR-shRNA-PC-3 and 5.57 ± 1.20 %ID/g in Lenti-SCR-shRNA-DU-145, P < 0.001) by PET quantification. Moreover, the volumes of prostate cancer xenograft tumors with knockdown of hCtr1 (179 ± 111 mm(3) for Lenti-hCtr1-shRNA-PC-3 or 39 ± 22 mm(3) for Lenti-hCtr1-shRNA-DU-145) were significantly smaller than those without knockdown of hCtr1 (536 ± 191 mm(3) for Lenti- SCR-shRNA-PC-3 or 208 ± 104 mm(3) for Lenti-SCR-shRNA-DU-145, P < 0.01). CONCLUSION: Overall, data indicated that hCtr1 is a promising theranostic target, which can be further developed for metabolic imaging of prostate cancer using (64)CuCl2 PET/CT and personalized cancer therapy targeting copper metabolism.

Potent anticancer activity of pyrrolidine dithiocarbamate-copper complex against cisplatin-resistant neuroblastoma cells.

Effective drugs are urgently needed for the treatment of advanced neuroblastoma refractory to conventional chemotherapy. Pyrrolidine dithiocarbamate (PDTC) is a copper-binding ligand, which showed cytotoxicity on many human tumor cells after binding with copper ions. In this study, we synthesized a copper-PDTC complex, which was characterized as a Cu(PDTC)2 complex, with elemental analyses (Fourier transform infrared, electrospray ionization mass spectra, and ultraviolet-visible spectroscopy). The Cu(PDTC)2 complex suppressed the proliferation of BE(2)C cells, a human neuroblastoma cell line, with an IC50 of 8.0 micromol/l, which was more potent than cisplatin (IC50 of 80 micromol/l). Treatment of BE(2)C cells with the Cu(PDTC)2 complex caused the S-phase arrest of cell cycle progression, cellular apoptosis, and necrosis, and increased the expression of p53 protein. The Cu(PDTC)2 complex holds potential as a new drug for the treatment of refractory neuroblastoma in children.

[Correlation between porcine CAST gene polymorphism with muscle fiber histological traits and carcass characteristics].

Three polymorphisms were identified in the sixth intron of the CAST gene by PCR-RFLP using enzymes Msp I, Hinf I and Rsa I in 45 Jinpi F2 pigs. However, only three genotypes AACCEE, BBDDFF, and ABCDEF were detected in those pigs and the genotype frequencies were 0.1778, 0.2222 and 0.6000, respectively. Longissimus dorsi muscles were moved for paraffin serial sections and stained with hematoxylin-eosin or myosin heavy chain (MHC) by immunohistochemistry respectively. Fiber cross-sectional area, fiber density, fiber diameter, the rate of MHC type I fiber and the carcass characteristics were recorded. Correlation analysis showed that the skeletal muscle fiber area and the eye muscle area of the BBDDFF individual were significantly higher than those of the ABCDEF individual (P < 0.05).

[The histological characteristics of longissimus dorsi and its correlation with restriction fragments polymorphism of calpastatin gene in f2 Jinghua x Pietrain crossbred pigs].

In order to evaluate the genotype of CAST gene and its relationship with the muscle histology and other postmortem traits. 158 Jinpi F2 pigs were electrically stunned and exsanguinated. The blood and muscle samples were collected, and both postmortem and meat traits were recorded. PCR-RFLP, PAS and myosin heavy chain immunohistochemistry were employed to explore the relationship between the genotypes and the muscle histology. Based on the PAS reactivity, the muscle fibers can be divided into three types: I, II-a and II-b. Myosin heavy chain immunohistochemistry could differentiated the fibers into slow fibers and fast fibers, the ratio of slow fibers and fast fibers is about 7.62% and 92.38% respectively. The amplification products of the region between 6th and 7th exon of CAST gene were digested by HinfI and discriminated 2 polymorphic sites (524bp and 632bp) in endonuclease map. Only 3 genotypes (AA,AB and BB) was distinguished out. The frequency of AA, AB and BB is 0.1795, 0.5897 and 0.2308 respectively. The frequency of A and B is 0.4743 and 0.5256 respectively. Different genotypes had no statistical significant effect on area, water holding capacity, pH value, and conductivity of longissimus dorsi muscle. The results revealed that the genotypes had a significant effect on the aspect ratio of muscle and showed a negative correlation with the percentage of intramuscular connective tissue.

Mouse extrahepatic hepatoma detected on MicroPET using copper (II)-64 chloride uptake mediated by endogenous mouse copper transporter 1.

PURPOSE: This study was conducted to assess the positron-emitting copper (II)-64 chloride ((64)CuCl(2)) as a probe for imaging mouse extrahepatic hepatoma expressing mouse copper transporter 1 (mCtr1) with positron emission tomography (PET). PROCEDURES: Following the intravenous administration of (64)CuCl(2), athymic mice bearing extrahepatic hepatoma grafts were subjected to whole-body static PET imaging with a Concorde microPET R4 tomograph. Upon completion of the imaging study, immunohistochemistry (IHC) study of mCtr1 was performed with postmortem tissues. RESULTS: The mouse extrahepatic hepatoma grafts were well visualized on static microPET images. Quantitative analysis demonstrated that the tracer concentration in the hepatoma was significantly higher than those in the soft tissue of the right shoulder opposite to the tumor site and the brain (p < 0.001). mCtr1 immunoreactivity in the hepatoma graft was approximately 70% of that in liver, whereas (64)CuCl(2) concentration in the graft was approximately 11% of the liver concentration. CONCLUSIONS: The extrahepatic mouse hepatoma grafts may be visualized by Cu-64 PET, taking advantage of the (64)CuCl(2) uptake mediated by the functional endogenous mCtr1.