腺病毒介导的秦川牛固醇调节元件结合蛋白2基因(SREBP2)过表达载体的构建与鉴定

固醇调节元件结合蛋白2（sterol regulatory element binding protein 2,SREBP2）属于碱性螺旋-环-螺旋亮氨酸拉链转录因子家族,在胆固醇的代谢过程中具有重要的调控作用。克隆秦川牛（Bos taurus）的SREBP2基因并构建相应腺病毒过表达载体,包装扩繁获得高滴度腺病毒,对于在细胞水平上研究SREBP2基因的功能和作用机制具有重要作用。本研究以秦川牛脂肪组织为实验材料,提取总RNA并反转录为cDNA,依据GenBank收录的牛的SREBP2基因（Accession No.NM₀01205600）mRNA序列设计引物,克隆出SREBP2基因编码区（coding sequence,CDS）全长。将SREBP2基因与穿梭载体连接构建pAdTrack-CMV-SREBP2表达载体,用PmeⅠ分别酶切线性化pAdTrack-CMV-SREBP2和空白对照pAdTrack-CMV载体并转化含有骨架载体pAdEasy-1的大肠杆菌（Escherichia coli）BJ5183感受态进行同源重组,得到腺病毒重组载体pAd-SREBP2和pAd-CMV,分别用PacⅠ酶切后胶回收DNA大片段,并将回收产物转染293A细胞包装得到腺病毒Ad-SREBP2和Ad-CMV,增殖并提高病毒滴度,得到高滴度病毒后,用绿色荧光蛋白（green fluorescent protein,GFP）标记法测定显示Ad-SREBP2和Ad-CMV的病毒滴度分别为7×108和1.3×109GFU/mL。将Ad-SREBP2和Ad-CMV腺病毒侵染秦川牛前体脂肪细胞检测病毒的有效性,实时定量PCR检测结果显示,侵染48h时SREBP2基因的表达量提高了102.3倍。本研究成功克隆了秦川牛的SREBP2基因,构建了病毒重组体,包装增殖获得了高滴度病毒,为在细胞水平上基因功能的研究提供了基础资料。     

The Effect of Cholesterol Addition,Buffer, and pH on Equine Sperm Stored at 5°C

Cooled stallion semen has a short viable life, which ranges with acceptable motility and viability from 24 up to 48 hours. The purpose of this study was to compare the effects of storage pH, the ability of three different zwitterionic buffers, and cholesterol-loaded cyclodextrins (CLC) to preserve the motility and integrity of stallion sperm cooled to 5°C for 48 hours. Fourteen ejaculates were collected and split to receive CLC or not (control group). After incubation, each sample was split into six subsamples and diluted in KMT extender containing 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), or 2-(N-morpholino)ethanesulfonic acid (MES) buffers, and the final pH was adjusted to either 7.0 or 6.6, totalizing 12 experimental groups as a function of CLC, buffer, and pH variables (2 × 3 × 2 factorial). The motility parameters and integrity of plasma and acrosome membranes (live cell index) were determined using computer-automated semen analysis and epifluorescence microscopy at 3, 6, 24, and 48 hours of cooling period. According to results, pH was not a significant source of variation for motility and live sperm over different cooling periods. However, samples diluted in BES exhibited higher progressive motility within 3 hours and higher percentages of total motile cells after 48 hours of incubation at 5°C (P < .05). After 24 hours of storage, CLC-treated sperm samples presented higher motility than control group (P < .05), and after 48 hours of incubation, CLC-treated sperm exhibited higher percentages of live, motile, and progressively motile sperms (P < .05). We inferred that equine semen diluted in KMT containing BES as buffer and CLC treatment improve the equine sperm survival during storage at 5°C for 48 hours.     

The relationship between feed intake behaviour with intramuscular fat, cholesterol and fatty acid composition in pork

The aim of this study was to investigate the phenotypic relationship between feed intake (FI) characteristics with intramuscular fat, cholesterol and fatty acid composition in pork. Data were available on 202 Duroc barrows. Intramuscular fatness was positively correlated with a higher percentage of saturated (SFA) and monounsaturated fatty acids (MUFA), a lower percentage of polyunsaturated (PUFA), omega-3 (ω3) and omega-6 (ω6) fatty acids, and a lower ω6/ω3 and P:S ratio (p < 0.05). Faster growth resulted in a higher level of SFA and a lower level of ω3 fatty acids (p < 0.05). Increased FI, residual feed intake (RFI) and feed intake time (TIME) and a decreased feed intake frequency (FREQ) resulted in an increase in MUFA, a decrease in PUFA, ω3 and ω6 fatty acids and a lower P:S ratio (p < 0.05). In addition, increased FI and RFI and a decreased FREQ resulted in an increase in SFA (p < 0.05). Most of the correlations were a result of a concomitant relationship with intramuscular fatness and disappeared after correction for that trait. Serum total, high-density lipoprotein and low-density lipoprotein cholesterol levels were positively correlated with SFA and MUFA, and negatively with PUFA, omega-3 and omega-6 fatty acids, and the P:S ratio (p < 0.05). These results have implications for programmes aimed at improving meat quality through selection.     

酶法制取丝素降胆固醇肽的工艺研究

[目的]利用废蚕丝制取具有降胆固醇的活性肽。[方法]考察不同水解度的丝素活性肽对胆固醇在胆汁胶束溶液中溶解度的抑制率,并以此作为碱性蛋白酶水解蚕丝蛋白的评价指标,采用单因素试验对碱性蛋白酶的水解工艺进行了探讨。[结果]其最佳工艺条件：产物浓度为20g/L,酶用最为0.4g/L,反应温度为40℃,pH为9.5。[结论]碱性蛋白酶水解丝素蛋白的水解度为16.5%时,产物具有最佳活性。     

Role of apolipoprotein E in cholesterol efflux mediated by ABCA1 and ABCG1

AIM:To investigate the role of apolipoprotein E(ApoE) in cholesterol efflux mediated by ATP-binding cassette transporter A1(ABCA1) and ATP-binding cassette transporter G1(ABCG1). METHODS:RAW 264.7 cells were seeded in either 6-well or 24-well plates, and then incubated with 20 mg/L low-density lipoprotein receptor gene knockout(LDLr^-/-) mouse lipoprotein 20 mg/L ApoE gene knockout(ApoE^-/-) mouse lipoprotein or culture medium alone. The changes of intracellular lipid content were measured by transmission electron microscopy and enzymatic colorimetric method. The cholesterol efflux was determined by liquid scintillation. The mRNA and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blotting, respectively. RESULTS:The ApoE^-/- mouse lipoprotein increased the content of intracellular cholesterol ester by 60% compared with the control cells. In addition, ApoE^-/- mouse lipoprotein treatment decreased the cholesterol efflux to apolipoprotein A-I(ApoA-I) and high-density lipoprotein(HDL) compared with LDLr^-/- mouse lipoprotein treatment. ApoE^-/- mouse lipoprotein treatment inhibited the mRNA and protein levels of ABCA1 and ABCG1 compared with LDLr^-/- mouse lipoprotein treatment. CONCLUSION:Apolipoprotein E plays an important role in the cholesterol efflux of macrophages, which is associated with its regulatory effect on the expression of ABCA1 and ABCG1.     

IMAGING DIAGNOSIS—MRI CHARACTERISTICS OF A FOURTH VENTRICULAR CHOLESTEROL GRANULOMA IN A DOG

A 2‐year‐old male American Bulldog experienced paroxysmal staggering, altered consciousness, and hyperesthesia. Magnetic resonance (MR) imaging enabled recognition of a fourth ventricular mass causing compression of the cerebellum and brainstem and obstructive hydrocephalus. The mass was uniformly T2‐hyperintense and predominantly T1‐hypointense. A fluid line was evident on the fluid‐attenuated inversion recovery images. A thin rim of contrast enhancement was noted. Histopathologic diagnosis was a cholesterol granuloma. We were unable to identify any other reports of a cholesterol granuloma residing in the fourth ventricle of a dog. This case report documents the clinical, diagnostic imaging, and histopathologic findings of a canine intracranial cholesterol granuloma. © 2012 Veterinary Radiology & Ultrasound.     

What is your diagnosis? Intracranial mass in a cat

Abstract: A 14‐year‐old female spayed cat was presented with a 3–4‐month history of circling to the left and intermittent head pressing. Neurologic examination findings localized the lesion to the left supratentorial region. Using magnetic resonance imaging, an extra‐axial mass was found on the dorsal aspect of the brain at the level of the frontal and parietal lobes, compressing and displacing the brain ventrally and caudally. Craniectomy was performed and the mass was submitted for cytologic and histopathologic evaluation. Impression smears revealed abundant cholesterol crystals and loose clusters of mildly pleomorphic spindle cells, compatible with a meningioma. The histopathologic diagnosis was meningioma with psammoma bodies and numerous cholesterol clefts. Abundant cholesterol crystals within meningiomas in cats may present a diagnostic challenge when nucleated cells are scant. Other differential diagnoses for abundant cholesterol crystals in an intracranial mass include cholesterol granulomas and keratinizing cysts.     

胆固醇转运蛋白NPCIL1的研究进展

NPCIL1是近年来人们研究高脂血症的重点内容，该蛋白已被证实在胆固醇的肠道吸收和胆汁分泌中发挥了关键作用。NPCIL1调节体内胆固醇的生物合成，是维持生物体胆回醇动态平衡的重要因素，同时也是新型降脂药物依泽替米贝的作用靶点。     

壳聚糖对奶牛血清胆固醇和蛋白质的影响

选择体重、胎次、泌乳期、产奶量相近、营养中等、临床健康、CMT检测呈阴性的中国荷斯坦奶牛40头,随机分为对照组和试验1、2、3组四个组。在基础日粮中分别添加壳聚糖0g/d、2g/d、4g/d、6g/d,连续饲喂5周。结果:试验1、2组血清胆固醇含量分别为2.75～3.07mmol/L、2.69～3.05mmol/L(P>0.05);试验3组牛在0～2周分别为2.75～3.03mmol/L(P>0.05),3～5周分别为2.31±0.62(mmol/L)、2.15±0.51(mmol/L)和2.06±0.49(mmol/L);分别降低了21.24%、21.82%和28.96%,差异显著(P<0.05)。血清总蛋白、白蛋白和球蛋白含量各组均无显著性差异(P>0.05)。结论:应用6g/d壳聚糖能够降低泌乳奶牛血清胆固醇的含量,但不影响奶牛血清蛋白质的含量。壳聚糖可以作为饲料添加剂在牛群中长期使用。     

鱼类低温适应机制的研究——Ⅱ.驯化温度对草鱼和鲮鱼肌肉线粒体膜脂肪酸组成和胆固醇含量的影响

采用气相色谱法和气相色谱/质谱联用法对不同温度驯养的草鱼和鲮鱼肌肉线粒体膜脂肪酸组成进行了分析。10℃适应的草鱼和25℃适应的草鱼相比,肌肉线粒体膜脂肪酸中不饱和脂肪酸/饱和脂肪酸(U/S)值明显升高,脂酰链平均长度也从25℃时的19.08个碳原子增加到10℃时的20.46个碳原子。而鲮鱼的脂肪酸组成在不同适应温度下的变化不明显。此外,还测出了不同驯养温度下草鱼和鲮鱼肌肉线粒体中胆固醇含量的变化,发现两种鱼的胆固醇含量在低温下都显著下降。作者认为,低温适应下的草鱼由于肌肉线粒体的脂肪酸组成和胆固醇含量都发生变化,因而其肌肉线粒体膜的流动性明显增加;而鲮鱼肌肉组织线粒体中胆固醇含量虽也减少,但脂肪酸组成变化甚微,因此,鲮鱼肌肉组织线粒体膜流动性增加很有限。这可能就是鲮鱼耐寒能力差的重要原因之一。