An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.     

烟叶脉坏死病毒原鉴定及cDNA克隆与序列分析

田间采集辽宁地区烟叶脉坏死病标样所得分离物,在测定的１２个科３５种植物中只侵染茄科的一些烟草品种及洋酸浆（ｐｈｙｓａｌｉｓｆｌｏｒｉｄａｎａ）,可由桃蚜（Ｍｙｚｕｓｐｅｒｓｉｃａｅ）传播。病叶汁液稀释限点（ＤＥＰ）为１０￣（－２）－v１０￣（－３）;失毒温度（ＴＩＰ）为５５一６０℃;体外保毒期（Ｌ）为４８－７２小时。病毒粒体形态呈线条状７２０×１２ｎｍ,病叶脉坏死部细胞质中含风轮状内含体。病毒提取物的紫外最大吸收为２６５ｎｍ,最小吸收为２４５ｎｍ,Ａ＿（２８０）／Ａ＿（２６０）为０．８２。该病毒分离物与ＰＶＹ￣０抗血清呈阳性反应。以病毒ＲＮＡ为模板,按国外报道的ＰＶＹ￣Ｎ序列合成引物经逆转录合成ｃＤＮＡ。用ＰＣＲ扩增出约０．８０ｋｂ的ＣＰ基因片段,将这一片段插入载体ｐＧＥＭ７Ｚ－ｆ（＋）中转化Ｅ．ｃｏｌｉＤＨ５ａ菌株得到了ＣＰ基因的克隆。ｃＤＮＡ序列分析表明,和国外报道的ＰＶＹ￣Ｎ序列同源性极高,初步表明引起辽宁地区烟叶脉坏死病的毒原为ＰＶＹ￣Ｎ。     

Effects of Irradiation on the Postnatal Development of the Brain in a Genetic Mouse Model of Globoid Cell Leukodystrophy

Irradiation is one way to condition Twitcher mice--a natural model of globoid cell leukodystrophy (GLD)--prior to receive bone marrow transplantation (BMT). BMT showed to delay but not to completely prevent GLD disease in treated mutants. The reasons why BMT is not completely preventive in Twitchers are unclear but we speculate that irradiation might contribute to worsen the neurological impairments generated by the disease by altering postnatal neurogenesis. To test this hypothesis, we examined proliferation, migration and differentiation of neural precursors in neurogenic areas of the Twitcher brain after exposure of 5 day-old mutant pups to 620 rad, a non-lethal dose that leads to 80-90% of bone-marrow engraftment in classic BMT. Twitchers showed to be sensitive to irradiation, leading to a severe retardation of body growth of irradiated mutants. Irradiated Twitchers had reduced proliferation of neural precursors and increased astrogliosis and microgliosis, with reduced numbers of migratory neuroblasts and significantly less brain myelination. These effects were accompanied by caspase-3 activation and appeared largely irreversible in the lifespan of the Twitcher. Our work confirms that exposure of the neonatal brain to irradiation conditions such as those performed prior to BMT, can lead to long-lasting alterations of postnatal neurogenesis and myelination, which might contribute to worsen the progression of disease in these myelin mutants and to reduce the success of BMT.     

人结合珠蛋白cDNA克隆及其在大肠杆菌中的表达和鉴定

目的：克隆人结合珠蛋白（haptoglobin,Hp）cDNA,并在大肠杆菌中表达和鉴定。方法：从Hela细胞中分离总RNA,采用RT-PCR方法获得人Hp cDNA,分别克隆至原核表达载体pET-32a和PGEX-4T-1,转化至大肠杆菌BL21,IPTG诱导表达,并进行SDS—PAGE及Western blot鉴定。结果：成功构建了高效原核表达质粒PET-32a-Hp和PGEX-4T1-Hp;Western印迹结果表明,经IPTG诱导,在大肠杆菌中表达了分子量约30kD和37kD的目的蛋白;表达产物经Ni2^＋-NTA离子交换树脂纯化,纯度〉90％。结论：在E．coli成功表达和纯化了人Hp融合蛋白,为进一步开发人Hp诊断试剂打下基础。     

正常人肝细胞cDNA文库的构建及应用分析

目的利用Gubler-Hoffman法构建了正常人肝细胞的cDNA文库以筛选肝细胞内部与乙肝病毒感染相关的基因。方法首先采用TRIzol法提取正常人肝细胞总RNA,纯化mRNA。逆转录合成单链cDNA,然后合成双链cDNA。用Spin Column回收0.4kb以上片段,然后与Vector pAP3neo进行连接,利用电刺激转化法导入E.coliDH10B,利用PCR法检测文库的重组效率。结果扩增后的文库重组率为93.3%。结论已经成功地构建了正常人肝组织的cDNA文库,该文库可用于筛选与乙肝相关的基因及用于基因芯片的制作。     

草鱼CYP1A和ACT基因cDNA片段的克隆与鉴定

以Trizol法分别提取BNF诱导和对照处理草鱼的肝组织总RNA并合成cDNA第一链,以此为模板利用1对ACT特异性引物和(8条)6对CYP1A简并引物进行扩增。结果显示,引物对F0-R0在对照和诱导草鱼中均扩增得到预期ACTcDNA片段,而引物对F4-R4在诱导草鱼中获得预期CYP1A cDNA产物。这两个cDNA片段分别进行克隆、测序和比对,BLAST结果表明草鱼ACTcDNA片段(800 bp)与GenBank中ACT基因(登录号M25013)同源性为99.1%,推导氨基酸序列同源性为99.2%;草鱼CYP1A cDNA片段(439 bp)与鲤鱼同源性最高,为92.5%,推导氨基酸同源性为96.6%。上述序列提交GenBank,获得登录号分别为DQ211096和DQ211095。通过Mega 3.1软件的Neighbor-joining程序对CYP基因的部分cDNA序列和氨基酸序列进行比对分析并绘制进化树,根据CYP1A部分蛋白的系统发育关系,在进化上可以将参与比对的真骨鱼划分为4个主要的分支。     

Cloning and sequencing of the porcine K-casein cDNA

cDNA clones encoding porcine kappa-casein were isolated and sequenced. The porcine kappa-casein cDNA is 851 bp in length and encodes a preprotein of 188 amino acids.     

Cloning and sequencing of the porcine lactoferrin cDNA

cDNA clones encoding the entire porcine lactoferrin protein were isolated and sequenced. The porcine lactoferrin cDNA sequence presented here is 2259bp in length and encodes a leader peptide of 19 amino acids and a mature protein of 684 amino acids. Comparisons with other lactoferrins indicate a single glycosylation site. The iron- and anion-binding sites, and the cysteine residues involved in disulphide bonds, are conserved between the lactoferrin proteins.     

Cloning and characterization of the human colipase cDNA

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a lambda gt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted protein sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH2-terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. In vitro translation of mRNA transcribed from the cDNA gave a protein of the expected molecular size that was processed by pancreatic microsomal membranes. Sequence analysis of the in vitro translation product after processing demonstrated signal peptide cleavage and the presence of a human procolipase, as exists in the pig and horse colipases. DNA blot analysis was consistent with the presence of a single gene for colipase. RNA blot analysis demonstrated tissue-specific expression of colipase mRNA in the pancreas. Thus, we report, for the first time, a cDNA for colipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular Cloning and Expression of cDNA for Murine Galactocerebrosidase and Mutation Analysis of the Twitcher Mouse,a Model of Krabbe's Disease

Abstract: The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.     

Recovery of Infectious Pariacoto Virus from cDNA Clones and Identification of Susceptible Cell Lines

Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly.     

Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The K_m values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg²⁺ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.The shikimate pathway is a major pathway in primary and secondary plant metabolism (Herrmann, 1995). It provides chorismate for the synthesis of the aromatic amino acids Phe, Tyr, and Trp, which are used in protein biosynthesis, but also serves as a precursor for a wide variety of aromatic substances (Herrmann, 1995; Weaver and Hermann, 1997; Fig. ​Fig.1a).1a). Chorismate is also the starting point of a biosynthetic pathway leading to phylloquinones (vitamin K₁) and anthraquinones (Poulsen and Verpoorte, 1991). The first committed step in this pathway is the conversion of chorismate into isochorismate, which is catalyzed by ICS (Poulsen and Verpoorte, 1991; Fig. ​Fig.1b).1b). Its substrate, chorismate, plays a pivotal role in the synthesis of shikimate-pathway-derived compounds, and its distribution over the various pathways is expected to be tightly regulated. Elicited cell cultures of Catharanthus roseus provide an example of the partitioning of chorismate. Concurrently, these cultures produce both Trp-derived indole alkaloids and DHBA (Moreno et al., 1994). In bacteria DHBA is synthesized from isochorismate (Young et al., 1969). Elicitation of C. roseus cell cultures with a fungal extract induces not only several enzymes of the indole alkaloid biosynthetic pathway (Pasquali et al., 1992) but also ICS (Moreno et al., 1994). Information concerning the expression and biochemical characteristics of the enzymes that compete for available chorismate (ICS, CM, and AS) may help us to understand the regulation of the distribution of this precursor over the various pathways. Such information is already available for CM (Eberhard et al., 1996) and AS (Poulsen et al., 1993; Bohlmann et al., 1995) but not for ICS. Figure 1a, Position of ICS in the plant metabolism. SA, Salicylic acid, OSB, o-succinylbenzoic acid. b, Reaction catalyzed by ICS.Isochorismate plays an important role in bacterial and plant metabolism as a precursor of o-succinylbenzoic acid, an intermediate in the biosynthesis of menaquinones (vitamin K₂) (Weische and Leistner, 1985) and phylloquinones (vitamin K₁; Poulsen and Verpoorte, 1991). In bacteria isochorismate is also a precursor of siderophores such as DHBA (Young et al., 1969), enterobactin (Walsh et al., 1990), amonabactin (Barghouthi et al., 1991), and salicylic acid (Serino et al., 1995). Although evidence from tobacco would indicate that salicylic acid in plants is derived from Phe via benzoic acid (Yalpani et al., 1993; Lee et al., 1995; Coquoz et al., 1998), it cannot be excluded that it is also synthesized from isochorismate. In the secondary metabolism of higher plants, isochorismate is a precursor for the biosynthesis of anthraquinones (Inoue et al., 1984; Sieweke and Leistner, 1992), naphthoquinones (Müller and Leistner, 1978), catalpalactone (Inouye et al., 1975), and certain alkaloids in orchids (Leete and Bodem, 1976).ICS was first extracted and partially purified from crude extracts of Aerobacter aerogenes (Young and Gibson, 1969). Later, ICS activity was detected in protein extracts of cell cultures from plants of the Rubiaceae, Celastraceae, and Apocynaceae families (Ledüc et al., 1991; Poulsen et al., 1991; Poulsen and Verpoorte, 1992). Genes encoding ICS have been cloned from bacteria such as Escherichia coli (Ozenberger et al., 1989), Pseudomonas aeruginosa (Serino et al., 1995), Aeromonas hydrophila (Barghouthi et al., 1991), Flavobacterium K_3–15 (Schaaf et al., 1993), Hemophilus influenzae (Fleischmann et al., 1995), and Bacillus subtilis (Rowland and Taber, 1996). Both E. coli and B. subtilis have two distinct ICS genes; one is involved in siderophore biosynthesis and the other is involved in menaquinone production (Daruwala et al., 1996, 1997; Müller et al., 1996; Rowland and Taber, 1996). The biochemical properties of the two ICS enzymes from E. coli are different (Daruwala et al., 1997; Liu et al., 1990). Sequence analysis has revealed that the bacterial ICS enzymes share homology with the chorismate-utilizing enzymes AS and p-aminobenzoate synthase, suggesting that they share a common evolutionary origin (Ozenberger et al., 1989).Much biochemical and molecular data concerning the shikimate pathway in plants have accumulated in recent years (Schmid and Amrhein, 1995; Weaver and Hermann, 1997), but relatively little work has been done on ICS from higher plants. The enzyme has been partially purified from Galium mollugo cell cultures (Ledüc et al., 1991, 1997), but purification of the ICS protein to homogeneity has remained elusive, probably because of instability of the enzyme.Our interests focus on the role of ICS in the regulation of chorismate partitioning over the various pathways. Furthermore, we studied ICS in C. roseus to gain insight into the biosynthesis of DHBA in higher plants (Moreno et al., 1994). In this paper we report the first purification, to our knowledge, of ICS to homogeneity from a plant source and the cloning of the corresponding cDNA.     

Hela细胞P16~(ink4)cDNA的克隆及序列分析

P16~(ink4)是CDK抑制蛋白,参与调控细胞G1期至S期的转换。目前发现P16`(ink4)基因损伤与多种肿瘤的发生、发展有关,可能是功能上非常重要的抑癌基因。为了研究该基因的功能,以及突变对该基因功能的影响,本文应用RT-PCR方法,从Hela细胞中克隆了P16~(ink4)cDNA。扩增得到556bp片段(包括引物两端酶切位点的16bp)克隆于M13载体,测定了其DNA序列。该序列包括了P16~(ink4)cDNA编码区全部471bp,以及3’端69bp序列。表明P16~(ink4)cDNA已成功地得到克隆。     

青岛地区茄白粉病的病原菌鉴定

在青岛地区的保护地和露地发现茄白粉病,通过形态学和分子生物学进行鉴定以及致病性测定。结果将青岛地区茄白粉病的病原鉴定为Podosphaera xanthii,本文为该菌在我国茄子上的首次报道。     

Cloning and expression of cryptochrome2 cDNA in the rat.

Cryptochromes (CRY) are blue-light photoreceptors that regulate the circadian rhythm in animals and plants. In mammals, two types of CRY are involved in the regulation of circadian rhythm, but rat cryptochromes have not yet been identified. Therefore, we isolated and characterized cry2 cDNA from the rat brain. The cloned rat cry2 cDNA consists of 2,131 nucleotides and has a single open-reading frame that encodes the rat CRY2 of 594 amino acids with start and stop codons. The deduced amino acid sequence of the rat CRY2 was 97% identical with that of mice and 93% with humans, but it showed a relatively low identity of 64% with that of zebrafish. It also exhibited a high homology (about 70%) with CRY1 of mice and humans. A Northern blot analysis showed that rat cry2 was expressed in all of the tissues examined. Rat cry2 was expressed at a relatively higher level in peripheral tissues than in the brain. In situ hybridization in the whole brain indicated that the strong signal of cry2 mRNA is mainly present in the suprachiasmatic nucleus (SCN) region, but very weak in other brain regions. Therefore, present results indicate that rat cry2 may function in circadian photoreception in the rat brain.