The Breeding of Mutant p53 and Neu/erb B2 Dual transgenic Mice: Both of the Genes were Over expressed Specifically in the Mammary Gland

为了研究乳腺癌发生与发展过程中突变型ｐ５３和Ｎｅｕ基因之间的相互作用,我们构建了ｐ５３／Ｎｅｕ双转基因鼠。用５只成年雄性Ｎｅｕ转基因鼠与１０只成年雌性ｐ５３转基因鼠交配,对１５０只雌性杂交后代鼠进行ｐ５３／Ｎｅｕ双转基因鼠的筛选。为此,本实验建立了ＰＣＲ一步鉴定法。即：在同一个反应管内能同时满足小鼠β酪蛋白基因、ｐ５３基因和Ｎｅｕ基因上三个片段（分别为０．５ｋｂ、１．２ｋｂ、１．７ｋｂ）的扩增反应者被断定为双转基因鼠（Ｆｉｇ．１）。传统的Ｓｏｕｔｈｅｒｎ分析（Ｆｉｇ．２）与ＰＣＲ一步法鉴定结果相吻合。证明：该法的准确率为１００％。进一步、随机取样,Ｎｏｒｔｈｅｒｎ分析（Ｆｉｇ．３）表明：双转基因鼠（２号）在哺乳笫二天即出现了ｐ５３基因的高表达。继而、在姓娠笫１０、１７天,哺乳笫１０、２１天也检测到该基因的表达。另外,免疫组化染色也检测到了ｐ５３和Ｎｅｕ蛋白（未展示结果）。     

A novel receptor-targeted gene delivery system for cancer gene therapy

Some growth factor receptors, such as insulin like growth factor Ⅰ and Ⅱ receptor (IGF Ⅰ R, IGF Ⅱ R) and epidermal growth factor receptor (EGF R), have been proved to be over-expressed in a variety of human cancers derived from different tissue origins. Based on this molecular alteration, a polypeptide conjugate gene delivery system was designed and synthesized. It contains three essential moieties: a ligand oligopeptide (LOP) for receptor recognition, a polycationic polypeptide (PCP) such as protamine (PA) or poly-L-lysine (PL) as a backbone for DNA binding and an endosome-releasing oligopeptide (EROP) such as influenza baenagglutinin oligopeptide (HA20) for endosomolysis. These components are covalently conjugated as LOP-PCP-HA20 or in the form of a mixture of LOP-PCP and HA20-PCP. A 14 amino acid E5 was designed and synthesized as LOP for IGF Ⅰ R and IGF Ⅱ R, and a 16 amino acid GE7 as LOP for EGF R. Both E5 and GE7 systems could form stable complex with the plasmid DNA as E5-PCP/DNA/PCP-HA20 a     

Spina bifida cystica and severe congenital bilateral talipes equinovarus in one twin of a monoamniotic pair: a case report

Background

Spina bifida and congenital talipes equinovarus (CTEV) are common congenital malformations which may occur together and increase morbidity. Monozygous twins are particularly at risk of these malformations and discordance in one type of malformation is typical. The occurrence of both spina bifida and CTEV in one twin of a monozygotic pair is rare.

Case presentation

A 22 year-old Cameroonian primigravida at 36 weeks of a twin gestation was received in our district hospital at the expulsive phase of labour on a background of sub-optimal antenatal care. A caesarean section indicated for cephalo-pelvic disproportion was performed and life monoamniotic male twins were extracted. The first twin was normal. The second twin had spina bifida cystica and severe bilateral CTEV. Routine postnatal care was ensured and at day 2 of life, the affected twin was evacuated to a tertiary hospital for proper management. He was later on reported dead from complications of hydrocephalus.

Conclusions

Spina bifida cystica with severe bilateral CTEV in one twin of a monoamniotic pair illustrates the complexity in the interplay of causal factors of these malformations even among monozygotic twins who are assumed to share similar genetic and environmental features. The occurrence and poor outcome of the malformations was probably potentiated by poor antenatal care. With postnatal diagnoses, a better outcome was difficult to secure even with prompt referral. Early prenatal diagnoses and appropriate counseling of parents are cardinal.

     

Detection and characterization of diverse alpha- and betacoronaviruses from bats in China

Bats have been implicated as important reservoir hosts of alpha- and betacoronaviruses. In this study, diverse coronaviruses (CoVs) were detected in 50 of 951 (positive rate 5.3%) intestinal specimens of eight bat species collected in four provinces and the Tibet Autonomous Region of China by pan-coronavirus RT-PCR screening. Based on 400-nt RNA-dependent RNA polymerase (RdRP) sequence analysis, eight belonged to genus Alphacoronavirus and 42 to Betacoronavirus. Among the 50 positive specimens, thirteen gave rise to CoV full-length RdRP gene amplification for further sequence comparison, of which three divergent sequences (two from a unreported province) were subjected to full genome sequencing. Two complete genomes of betacoronaviruses (JTMC15 and JPDB144) and one nearly-complete genome of alphacoronavirus (JTAC2) were sequenced and their genomic organization predicted. The present study has identified additional numbers of genetically diverse bat-borne coronaviruses with a wide distribution in China. Two new species of bat CoV, identified through sequence comparison and phylogenetic analysis, are proposed.

Open image in new window

     

Effects of water gradients on soil enzyme activity and active organic carbon composition under Carex lasiocarpa marsh

The responses of soil enzyme activity of freshwater marsh, microbial biomass carbon (MBC), dissolved organic carbon (DOC) and aboveground biomass to water gradients were studied with Carex lasiocarpa pot culture experiment. The relationships between soil enzyme activity and MBC, DOC and aboveground biomass were discussed. The water gradients were W1, 15 cm; W2, ?5 cm; W3, ?5–5 cm; W4, submerged. The results indicated that acid phosphatase, invertase and urease activities were decreased with the increase of water level, while catalase activity was increased with moisture content increasing. Drying-wetting alternation (W3) increased soil enzyme activities if compared with W1. MBC content followed the order of W3 > W1 > W2 > W4, and the activities of invertase, urease and catalase were significantly positively correlated with MBC (p < 0.05). DOC content presented the order of W4 > W1 > W3 > W2, and the activities of urease and acid phosphatase were most significantly negatively correlated with DOC (p < 0.01). In addition, drying-wetting alternation promoted the growth of Carex lasiocarpa. When water submerged plants, the growth of Carex lasiocarpa was significantly inhibited. The aboveground biomass was positively related to soil enzyme activities. There were close relationships between the activities of invertase, urease and catalase and the growth situation of Carex lasiocarpa.     

长白山地区蚱属一新种(直翅目,蚱总科)

报道采自长白山地区蚱属昆虫一新种,长白山蚱Tetrix changbaishanensis sp．nov．。模式标本保存在东北师范大学生命科学学院动物标本室。     

Developmental Change of Sialidase Neu4 Expression in Murine Brain and Its Involvement in the Regulation of Neuronal Cell Differentiation

Sialidase Neu4 is reported to be dominantly expressed in the mouse brain, but its functional significance is not fully understood. We previously demonstrated that sialidase Neu3, also rich in mouse brain, is up-regulated during neuronal differentiation with involvement in acceleration of neurite formation. To elucidate physiological functions of Neu4, as well as Neu3, we determined expression during mouse brain development by quantitative RT-PCR. Expression was relatively low in the embryonic stage and then rapidly increased at 3–14 days after birth, whereas Neu3 demonstrated high levels in the embryonic stage and down-regulation after birth. Murine Neu4 was found to possess two isoforms differing in expression levels, developmental pattern, and enzymatic character. Distinct from the human isoforms, the murine forms, to a different extent, both catalyzed the removal of sialic acid from gangliosides as well as glycoproteins, and one isoform seemed to act on polysialylated NCAM efficiently, despite the low activity toward ordinary substrates. In situ hybridization demonstrated Neu4 mRNA to be present mainly in the hippocampus in which NCAM is rich and decreases after birth. During retinoic acid-induced differentiation, Neu4 expression was down-regulated in Neuro2a cells. Overexpression of Neu4 resulted in suppression of neurite formation, and its knockdown showed the acceleration. Thin layer chromatography of the glycolipids from Neu4-transfected cells showed ganglioside compositions to be only slightly affected, although lectin blot analysis revealed increased binding to Ricinus communis agglutinin (RCA) lectin of a ∼95-kDa glycoprotein, which decreased with cell differentiation. These results suggest that mouse Neu4 plays an important regulatory role in neurite formation, possibly through desialylation of glycoproteins.Sialidases catalyze the removal of sialic acid from non-reducing ends of glycoproteins and glycolipids. In mammals, four types of sialidases have so far been cloned, classified according to their subcellular localization and enzymatic properties (abbreviated to Neu1, Neu2, Neu3, and Neu4) (1–3). Studies have provided strong evidence that these sialidases play crucial roles in various physiological functions such as cell differentiation, cell growth, and malignant transformation. Among these sialidases, Neu4 is unique in its tissue expression pattern and enzymatic properties. In the mouse, it is dominantly expressed in brain, but its sialidase activity is very weak compared with other mouse sialidases (4). In contrast, human NEU4 is expressed not only in brain, but also in liver, kidney, and colon (5–7). We have demonstrated that NEU4 has two isoforms, differing in the N-terminal 12-amino acid residues that act as a mitochondrial-targeting sequence (7). Except for the subcellular localization, enzymatic properties are very similar. The short form of NEU4 (NEU4S) suppresses malignancy in colon cancer cells, mainly through desialylation of some glycoproteins, whereas the long form of NEU4 (NEU4L) may be involved in apoptosis with hydrolysis of ganglioside GD3 in mitochondria (8). Recently, Neu4 knockout mice (Neu4^−/−) were generated for pathological analysis (9). Neu4^−/− grew normally with a normal lifespan and proved fertile, but vacuolization of the lung and spleen was observed with a lysosomal storage phenotype, and the GM1/GD1a ratio was decreased in the brain. The observations on Neu4^−/− are very interesting, but there is some ambiguity in the available previous reports, because, as mentioned above, mouse Neu4 has been reported to have weak sialidase activity in vitro, and its expression is restricted in brain. To clarify this ambiguity and further understand the physiological functions of Neu4, we examined expression in the mouse brain and observed a possible involvement in neural differentiation in connection with another sialidase, Neu3, which greatly increases during differentiation of neuroblastoma cells (10, 11) and causes acceleration of neurite formation (10–13).In the GenBank^TM data base, nucleotide sequences of mouse Neu4 have been submitted as {"type":"entrez-nucleotide","attrs":{"text":"AY258421","term_id":"32402573","term_text":"AY258421"}}AY258421 and {"type":"entrez-nucleotide","attrs":{"text":"AK034236","term_id":"26329804","term_text":"AK034236"}}AK034236. The former contains a complete coding sequence of 1506 bp, with two ATGs at positions 1 and 70, and {"type":"entrez-nucleotide","attrs":{"text":"AK034236","term_id":"26329804","term_text":"AK034236"}}AK034236 encodes only the second ATG (4). The gene from {"type":"entrez-nucleotide","attrs":{"text":"AY258421","term_id":"32402573","term_text":"AY258421"}}AY258421 has been reported to encode Neu4, showing weak sialidase activity, but there is no information on whether the gene based on {"type":"entrez-nucleotide","attrs":{"text":"AK034236","term_id":"26329804","term_text":"AK034236"}}AK034236 encodes Neu4 with sialidase activity toward natural substrates. We have now extended our studies to the existence of different mouse Neu4 isoforms, focusing on their significance in neuronal cells by measuring expression levels during cell differentiation. We present, here, evidence that two murine Neu4 isoforms contribute to neurite formation.     

干旱年份三江平原沼泽甲烷(CH4)排放及影响因子

为了研究淡水沼泽CH4排放的影响因子及其机制,在中国东北部的三江平原样地观测了CH4排放通量以及部分影响因子.结果表明,在2003年4～10月生长季内,毛果苔草沼泽CH4通量平均值为3.30 mg·m-2·h-1(最小值为0.65 mg·m-2·h-1,最大值为13.47 mg·m-2·h-1),低于小叶章沼泽化草甸CH4通量平均值4.91 mg·m-2·h-1(最小值为-0.12 mg·m-2·h-1,最大值为16.25 mg·m-2·h-1).在不同阶段,两种类型沼泽CH4通量有明显的变化,控制因子也不一样.4月份低温限制了CH4通量,两种类型沼泽差异很小.5～7月下旬,小叶章沼泽化草甸CH4通量显著高于毛果苔草沼泽CH4通量(P＜0.01),这种差异是由融冻作用造成的.8月份,植株密度和剖面中活性有机碳的浓度成为主要的影响因子,毛果苔草沼泽CH4通量比小叶章沼泽化草甸高.     

An ER Complex of ODR-4 and ODR-8/Ufm1 Specific Protease 2 Promotes GPCR Maturation by a Ufm1-Independent Mechanism

Despite the importance of G-protein coupled receptors (GPCRs) their biogenesis is poorly understood. Like vertebrates, C. elegans uses a large family of GPCRs as chemoreceptors. A subset of these receptors, such as ODR-10, requires the odr-4 and odr-8 genes to be appropriately localized to sensory cilia. The odr-4 gene encodes a conserved tail-anchored transmembrane protein; the molecular identity of odr-8 is unknown. Here, we show that odr-8 encodes the C. elegans ortholog of Ufm1-specific protease 2 (UfSP2). UfSPs are cysteine proteases identified biochemically by their ability to liberate the ubiquitin-like modifier Ufm1 from its pro-form and protein conjugates. ODR-8/UfSP2 and ODR-4 are expressed in the same set of twelve chemosensory neurons, and physically interact at the ER membrane. ODR-4 also binds ODR-10, suggesting that an ODR-4/ODR-8 complex promotes GPCR folding, maturation, or export from the ER. The physical interaction between human ODR4 and UfSP2 suggests that this complex''s role in GPCR biogenesis may be evolutionarily conserved. Unexpectedly, mutant versions of ODR-8/UfSP2 lacking catalytic residues required for protease activity can rescue all odr-8 mutant phenotypes tested. Moreover, deleting C. elegans ufm-1 does not alter chemoreceptor traffic to cilia, either in wild type or in odr-8 mutants. Thus, UfSP2 proteins have protease- and Ufm1-independent functions in GPCR biogenesis.