普通烟草β-胡萝卜素羟化酶2基因克隆与生物信息学分析

β-胡萝卜素羟化酶(beta-carotene hydroxylase,BCH)是植物类胡萝卜素合成代谢中的关键限速酶,为获得烟草β-胡萝卜素羟化酶2(NtBCH2)基因序列,采用同源克隆法从烟草中分离NtBCH2的基因组DNA序列,基因登录号为JX101477。结果表明:1)同源克隆法从烟草中克隆出NtBCH2基因,NtBCH2基因组DNA全长2131bp,cDNA为1 083bp,含7个外显子和6个内含子。2)NtBCH2蛋白有309个氨基酸,分子量为34.79kD,具有7个糖基化位点,11个磷酸化位点,4个跨膜域。3)系统发育树与BLAST分析表明,NtBCH2是番茄的SlBCH和辣椒CaBCH2的同源基因;GENEVESTIGATOR数据库芯片结果表明,NtBCH2在幼嫩的茎和子叶中表达量最高。     

A retrospective review of treatment and response of high‐risk mast cell tumours in dogs

This retrospective case series evaluates survival outcome of 94 dogs with high metastatic risk mast cell tumours (MCT). Patients were treated with a cytotoxic chemotherapy protocol or the tyrosine kinase inhibitor masitinib, in the presence of gross disease or as an adjunct to surgical resection of the primary tumour. In patients presenting with metastatic disease, surgical resection of the primary tumour with adjunctive therapy with any chemotherapy incurred a significant survival advantage [median survival time (MST): 278 days] compared to patients receiving chemotherapy without surgical excision of the primary tumour (MST: 91 days, P < 0.0001). Patients with a surgically excised Patnaik grade II tumour and high Ki‐67 in the absence of metastatic disease treated with vinblastine and prednisolone showed a significantly longer survival (MST: 1946 days) than those treated with masitinib (MST: 369 days, P = 0.0037). Further prospective case‐controlled clinical trials of high‐risk MCTs are required to make precise evidence‐based treatment decisions for individual patients.     

Ascorbic acid status as affected by dietary treatment in the Siberian sturgeon (Acipenser baeri Brandt): tissue concentration,mobilisation and L-gulonolactone oxidase activity

A study was conducted to evaluate tissue storage and mobilisation of L-ascorbic acid (AA) in the Siberian sturgeon (Acipenser baeri) fed three different experimental diets. The three treatments consisted of a diet devoid of vitamin C (diet A0) and two diets supplemented with equivalent of 300 mg AA kg^–1 in the form of either silicone-coated ascorbic acid (diet SC) or of ascorbyl-2-polyphosphate (diet AP). During the first phase (4 months) of the trial, six batches of 130 Siberian sturgeon (initial body weight: 25.5±0.5 g) each were fed one of the three diets in duplicate. During the second phase (3 months), fish from groups SC and AP were switched to diet A0 and those fed diet A0 during the first phase were switched to diet SC. Irrespective of the dietary treatment, growth rates were not significantly different from each other. At the end of phase I, in all tissues studied, total ascorbic acid (TAA) concentrations were higher in Siberian sturgeon fed diet AP than in the other two groups. During phase II, tissue ascorbate depletion was also higher in the AP group than in the other two groups. Transfer of the AA-free diet fed group onto a diet supplemented with 300 mg AA kg^–1 (diet SC) led to a slight increase in the TAA concentrations in all tissues. Blood plasma tyrosine concentrations were not significantly different between the three groups. Whole-body collagen levels were affected by dietary AA levels or forms at the end of phase I; the differences were not significant at the end of phase II. Muscle collagen levels were slightly affected. L-Gulonolactone oxidase activity was found in the kidney of Siberian sturgeon, but not in the liver. The ascorbyl-2-polyphosphate appears to be either better utilised by Siberian sturgeon, like in many other teleosts, or more stable than the silicone-coated AA during food processing and storage. Presence of L-gulonolactone oxidase activity in Siberian sturgeon kidney combined with the absence of gross scorbutic signs in AA-free diet fed groups expressing very good growth rates suggested no need of dietary AA byA. baeri.     

Insulin and IGF-I receptors and tyrosine kinase activity in carp ovaries: changes with reproductive cycle

Insulin and insulin-like growth factor I (IGF-I) receptors from carp ovaries were semipurified with wheat germ agglutinin at different moments of the reproductive cycle and their binding characteristics and tyrosine kinase activity were studied. Specific receptors for insulin and IGF-I were found. IGF-I receptors presented higher binding (23.8 ± 1.5%), number of receptors (965 ± 20fm/mg) and affinity (K_D 0.24 ± 0.03nM) than those shown for insulin receptors (4.1 ± 1%, 530 ± 85fm/mg and 0.85 ± 0.1nM, respectively). Insulin and IGF-I receptors have a tyrosine kinase activity which is not different from that found in muscle of the same species. Seasonal changes were found in binding, with maximum values for insulin and IGF-I reached at the end of pre-spawning period (June). However, while IGF-I binding was observed in all stages, insulin binding decreased in autumn and disappeared in winter, which suggests a different role for the two peptides in ovarian physiology.     

甘蓝型油菜烷羟化酶基因MAH1的克隆与表达分析

在植物蜡质合成途径中,中链烷烃羟化酶(mid-chain alkane hydroxylase,MAH)催化烷烃羟基化形成二级醇,进一步氧化为酮。本研究以拟南芥P450依赖性酶CYP96A15/MAH1基因为探针,采用电子克隆与RT-PCR技术,获得2个甘蓝型油菜MAH1的全长编码区序列,分别命名为BnMAH1-1和BnMAH1-2(Gen Bank登录号分别为KT795344和KT795345)。二者ORF长度均为1491 bp,无内含子,核苷酸与氨基酸序列分别有92.4%与90.9%的一致性。根据编码区预测的BnMAH1-1和BnMAH1-2前体蛋白均为包含496个氨基酸残基的多肽链,具有典型的P450蛋白家族保守结构P415x R417x、K螺旋(E359xx R362)、C末端的血红素结合域(F436xx Gx Rx Cx G445)以及氧结合带保守区域(A/G)G309x(D/E)T312(T/S)。NCBI Blast N、氨基酸序列多重比对与系统学分析表明,两者与拟南芥MAH1/CYP96A15同源性最高。实时荧光定量PCR表明,BnMAH1-1与BnMAH1-2主要在甘蓝型油菜茎、叶、花、及角果中表达,其中在叶片中的表达量最高,在根系中的表达量很低,这与角质层蜡质主要沉积在植株地上部分相一致。BnMAH1-1和BnMAH1-2在无蜡粉材料茎、叶片中几乎不表达,表明蜡质的减少与MAH1的转录下调有关。BnMAH1-1与BnMAH1-2受SA、Me JA、ACC、ABA、Na Cl及干旱胁迫诱导表达,其中BnMAH1-1可能在水分胁迫响应中起主要作用。     

Tyrosine Ameliorates Some of the Clinical,Biochemical and Haematological Effects of Acute Stress Associated with Transportation of Desert Sheep

We studied some clinical, biochemical and haematological variables in Desert (Najdi) sheep acutely stressed in the course of individual road transportation, and the influence thereon of pretreatment with tyrosine. Transportation for 30 min resulted in variable but statistically insignificant increases in heart, pulse and respiratory rates. It also caused significant increases in the plasma concentration of cortisol (from 43.5 to 101.7 mmol/L) and glucose (from 3.1 to 4.5 mmol/L), and a decrease in that of magnesium (from 0.85 to 0.72 mmol/L). The endogenous thiocyanate level was unaffected. The transportation stress also decreased the haematocrit (PCV) and the number of lymphocytes, and increased the concentration of haemoglobin. Pretreatment of sheep with tyrosine at a dose of 100 mg/kg by the intravenous route significantly ameliorated the stress-induced clinical, biochemical and haematological changes. The treatment caused no overt adverse effects.     

Immunohistochemical analysis of pseudorabies virus spread through neurons innervating the eyeball

Pseudorabies virus (PRV) was inoculated intraocularly into BALB/c mice, and the distribution pattern of cells positive for several neurotransmitters and viral antigens in the eyeball, trigeminal nerve ganglia, and superior cervical ganglia was examined immunohistochemically to clarify the neural route of the virus spread. In the eyeball, substance P (SP)- and calcitonin gene-related peptide (CGRP)-positive cells were detected in the ipsilateral iris and ciliary body, neuropeptide tyrosine (NPY)-positive cells were detected in the choloid membrane, and tyrosine hydroxylase (TH)-positive cells were detected in the ipsilateral inner nuclear layer of the retina; all these cells contained viral antigens. In the superior cervical ganglia, viral antigen-positive cells containing TH or NPY were found at bilateral sites. In the trigeminal nerve ganglia, viral antigen-positive cells containing SP or CGRP were found at bilateral sites. These findings indicated that the SP- and CGRP-positive ganglion cells of the trigeminal nerve ganglia innervating the iris or ciliary body, and the NPY-positive ganglion cells of the superior cervical ganglia innervating the iris, ciliary body, and choroid membrane served as the route for the virus spread. These findings also suggested that dopaminergic neurons, such as the TH-positive retinal cells and TH-positive ganglion cells of the superior cervical ganglia, served as the route for virus spread.     

家蚕核型多角体病毒酪氨酸蛋白磷酸酯酶基因核苷酸序列分析

从家蚕核型多角体病毒中国镇江株 (BmNPV ZJ)基因组DNA中克隆出酪氨酸蛋白磷酸酯酶基因 (ptp) ,该基因的编码部分由 5 0 7个核苷酸组成 ,其中A为 16 3、C为 99、G为 113、T为 132 ,G +C含量约为 4 2 %。根据其核苷酸序列推演的蛋白质由 16 8个氨基酸残基组成 ,其中含有酪氨酸蛋白磷酸酯酶催化活性区的 11个氨基酸“HC”基序。该基因与苜蓿银纹夜蛾核型多角体病毒 (AcMNPV)ptp和BmNPV T3株 (日本 )的ptp核苷酸序列的同源性分别为 96 8%和 98 2 %。BmNPV ZJ酪氨酸蛋白磷酸酯酶 (BmNPV ZJPTPase)的氨基酸全序列与AcMNPV、BmNPV T3、芹菜夜蛾核型多角体病毒 (AfMNPV)PTPase和黄杉毒蛾核型多角体病毒 (OpMNPV)PTPase 1的氨基酸全序列的同源性分别为 97%、97 6 %、96 %和 6 0 % ,而与OpMNPVPTPase 2的同源性仅为 2 0 %。在NCBI数据库中查找BmNPV ZJPTPase的同源性序列 ,查找到的 5 99381个序列中发现至少有 14种mRNA加帽酶其N端部分存在PTPase催化活性区的“HC”基序 ,但其氨基酸全序列的同源性只有 31%～ 32 %。该基因序列已被GenBank数据库收录 ,登录号为AF316 871     

Expression of receptor tyrosine kinases VEGFR-1 (FLT-1), VEGFR-2 (KDR), EGFR-1, PDGFRa and c-Met in canine primary brain tumours

Inhibition of tumour growth and angiogenesis by targeting key growth factor receptors is a promising therapeutic strategy for central nervous system tumours. Characterization of these growth factor receptors in canine primary brain tumours has not been done. Using quantitative real‐time TaqMan polymerase chain reaction (PCR), we evaluated the expression of messenger RNA (mRNA) for five tyrosine kinase growth factor receptors (vascular endothelial growth factor receptor [VEGFR]‐1, VEGFR‐2, endothelial growth factor receptor [EGFR]‐1, platelet‐derived growth factor receptor a [PDGFRa], and c‐Met) relative to normal cerebral cortex in 66 spontaneous canine primary brain tumours. Increased expression of VEGFR‐1 and VEGFR‐2 mRNA was greatest in grade IV astrocytomas (glioblastoma multiforme) and grade III (anaplastic) oligodendrogliomas. EGFR‐1 mRNA expression was more consistently increased than the other receptors in all tumour types, while increased PDGFRa mRNA expression was mostly restricted to oligodendrogliomas. The similarities in increased expression of these tyrosine kinase growth factor receptors in these canine tumours, as compared to data from their human counterparts, suggest that common molecular mechanisms may be present.