The Mechanism of CSF-1-induced Wiskott-Aldrich Syndrome Protein Activation in Vivo: A ROLE FOR PHOSPHATIDYLINOSITOL 3-KINASE AND Cdc42*

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.Macrophages, terminally differentiated cells of the mononuclear phagocytic lineage, are found throughout the body and play important roles in normal tissue development and immune defense. However, in certain circumstances, excessive recruitment of macrophages has been shown to participate in the progression of several diseases, inflammatory (rheumatoid arthritis) or metabolic (atherosclerosis), as well as in tumor progression (1–3). Importantly expression of colony-stimulating factor-1 (CSF-1),⁴ the most pleiotropic macrophage growth factor, has been correlated with the progression of these disease states (for a review, see Ref. 4). Inhibition of undesirable macrophage recruitment to specific sites in response to CSF-1 is therefore an attractive goal for therapies (5).In addition to stimulating survival, proliferation, and differentiation of monocytes and macrophages, CSF-1 is also a potent chemotactic factor inducing the migration of these cell types (for a review, see Ref. 4). CSF-1 stimulation leads to the rapid production of F-actin-rich protrusions and the spreading and migration of macrophages (4). All CSF-1 effects are mediated through its tyrosine kinase receptor (CSF-1R), which upon activation leads to phosphorylation of tyrosine residues in a number of signaling molecules. Downstream molecules essential for macrophage migration in response to CSF-1 include phosphatidylinositol 3-kinase (PI3K) isoforms β and δ (6, 7). PI3K may potentially regulate migration through the activation of guanine nucleotide exchange factor activity to Rac1 and Cdc42, which are required for CSF-1-elicited protrusions (8, 9) and chemotaxis (10). The major means by which Rac and Cdc42 regulate the Arp2/3 complex is through the Wiskott-Aldrich syndrome protein/Wiskott-Aldrich syndrome verprolin-homologous (WASP/WAVE) family of proteins (11). A Rac1-IRSp53-Abi1-WAVE2 complex has been shown to mediate CSF-1-induced macrophage motility (12, 13), and a unique role for WASP in macrophage chemotaxis to CSF-1, formylmethionylleucylphenylalanine, MCP-1, and MIP-1α has been demonstrated (14, 15). WASP is a hematopoietic cell-specific regulator of Arp2/3-dependent actin remodeling. The catalytically active domain of WASP lies in its C terminus, which is conserved among all WASP/WAVE proteins and contains a VCA (verprolin homology, cofilin-like, and acidic region) domain capable of activating the Arp2/3 complex. The other domains found in WASP can regulate, directly or indirectly, the activity of its VCA domain (for a review, see Ref. 16). Both WASP and N-WASP bind activated Cdc42 through their GTPase-binding domain, which is believed to cause a structural transition that results in dissociation of the intramolecular contacts leaving the VCA domain accessible for Arp2/3 binding (17, 18). In addition, biochemical studies have revealed that several signaling molecules, including WASP-interacting SH3 protein, WASP-interacting protein, Grb2, phosphoinositides, and Src family kinases, activate N-WASP (for reviews, see Refs. 16 and 19). Phosphorylation of WASP has also been proposed to activate Arp2/3-mediated actin polymerization in vitro (20–22).Recently different probes have been developed that detect a conformational change in N-WASP and therefore reflect its activation (23–25). Using either a fluorescence resonance energy transfer (FRET)-based biosensor that detects a conformational change in N-WASP (23, 24) or antibodies that can only bind to the open conformation of N-WASP (25), N-WASP has been shown to be activated in response to epidermal growth factor in HEK293 cells and in MTLn3 carcinoma cells. This activity has been temporally localized to subcellular compartments important for carcinoma cell chemotaxis and invasion (24). We have adapted these approaches to explore the signal transduction pathways responsible for the activation of WASP in vivo.     

非酒精性脂肪肝大鼠PPARα基因表达及脂代谢和胰岛素水平的变化

目的观察非酒精性脂肪肝（NAFLD）大鼠肝组织中PPARα基因的表达,并用PPARct激动剂进行干预,探讨其与胰岛素抵抗、脂代谢紊乱的关系。方法大鼠随机分为①正常对照组、②高脂模型组、③PPARα激动剂干预组,利用高脂饮食建立大鼠非酒精性脂肪肝模型。12周后,检测大鼠血脂、肝功能、血糖、胰岛素水平及胰岛素抵抗指数;RT-PCR法分析PPARα基因的表达;观察肝脏的形态学改变。结果PPARa激动剂可降低NAFLD大鼠转氨酶、血脂水平及胰岛素抵抗指数,可促进NAFLD大鼠中PPARa基因的表达;肝脏形态学明显改善。结论PPARα激动剂能改善NAFLD大鼠脂质代谢紊乱,有明显的保肝降酶作用,具有适度的胰岛素增敏作用。PPARα及其配体在NAFLD发病机制及治疗中的进一步深入研究,将为临床防治NAFLD提供新的思路。     

SD雌性大鼠性发育早期性器官等脏器和性激素的动态变化

目的探讨正常SD雌性大鼠性成熟前不同日龄段的脏器与促黄体生成素（LH）、促卵泡素（FSH）、雌二醇（E2）等性激素的变化及其关系。方法从生产群中取出60窝密度状态一致的SD大鼠,在不同日龄随机选取雌性大鼠,检测15、25、32、40日龄时大鼠体重、主要脏器指数,子宫、卵巢组织变化和15、25、32、40、60日龄大鼠血清LH、FSH、E2水平。结果记录了SD雌性大鼠性成熟前各脏器指数和卵巢、子宫组织变化,结果显示大鼠卵巢、子宫的增长速度大于体重的增长,而其他脏器增速大都小于体重的增长。本研究还记录了血清LH、FSH、E2水平在不同日龄段的变化规律,表明血清LH、E2浓度在32日龄时出现较为明显升高。结论不同日龄大鼠脏器指数的动态变化提示大鼠性器官在性发育早期得到机体的优先发育。血清LH、E2水平在32日龄时有了明显升高,提示性腺轴功能已经激活。60日龄大鼠血清性激素水平的波动类似于动情周期的规律性变化,推测大鼠在60日龄前即已进入性成熟,这些结果将为大鼠性发育的相关研究提供重要的参考数据。     

Developing human laboratory models of smoking lapse behavior for medication screening

Use of human laboratory analogues of smoking behavior can provide an efficient, cost-effective mechanistic evaluation of a medication signal on smoking behavior, with the result of facilitating translational work in medications development. Although a number of human laboratory models exist to investigate various aspects of smoking behavior and nicotine dependence phenomena, none have yet modeled smoking lapse behavior. The first instance of smoking during a quit attempt (i.e. smoking lapse) is highly predictive of relapse and represents an important target for medications development. Focusing on an abstinence outcome is critical for medication screening as the US Food and Drug Administration approval for cessation medications is contingent on demonstrating effects on smoking abstinence. This paper outlines a three-stage process for the development of a smoking lapse model for the purpose of medication screening. The smoking lapse paradigm models two critical features of lapse behavior: the ability to resist the first cigarette and subsequent ad libitum smoking. Within the context of the model, smokers are first exposed to known precipitants of smoking relapse (e.g. nicotine deprivation, alcohol, stress), and then presented their preferred brand of cigarettes. Their ability to resist smoking is then modeled and once smokers 'give in' and decide to smoke, they participate in a tobacco self-administration session. Ongoing and completed work developing and validating these models for the purpose of medication screening is discussed.     

Detection of Nonstructural Protein 6 in Murine Coronavirus-Infected Cells and Analysis of the Transmembrane Topology by Using Bioinformatics and Molecular Approaches

Coronaviruses encode large replicase polyproteins which are proteolytically processed by viral proteases to generate mature nonstructural proteins (nsps) that form the viral replication complex. Mouse hepatitis virus (MHV) replicase products nsp3, nsp4, and nsp6 are predicted to act as membrane anchors during assembly of the viral replication complexes. We report the first antibody-mediated Western blot detection of nsp6 from MHV-infected cells. The nsp6-specific peptide antiserum detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6 products of approximately 23 and 25 kDa. Analysis of nsp6 transmembrane topology revealed six membrane-spanning segments and a conserved hydrophobic domain in the C-terminal cytosolic tail.Coronaviruses are enveloped, positive-stranded RNA viruses that sequester host cell membranes to assemble viral replication complexes in the cytoplasm of infected cells (2, 21). In the case of murine coronavirus mouse hepatitis virus (MHV), three viral proteases process the replicase polyproteins (3, 4, 5, 9, 12, 13, 14, 16, 18, 19, 24, 26) into intermediates and 16 mature nonstructural protein (nsp) products (Fig. ​(Fig.1A).1A). It is unclear whether the intermediate forms or the mature nsps are responsible for assembly of the viral replication complex. The replicase proteins nsp3, nsp4, and nsp6 contain transmembrane (TM)-spanning sequences that are proposed to be important for sequestering endoplasmic reticulum (ER) membranes to form the double-membrane vesicles which are the site of viral RNA synthesis (11, 17). However, the mechanism used by the nsps to generate double-membrane vesicles is not yet understood. Recent reports (8, 15, 22, 23, 28) and the study presented here have unraveled the membrane topology of these nsps. nsp4 is a glycoprotein with four TM domains (8, 22, 23, 28). nsp3 anchors its 213-kDa multidomain protein to ER membranes, likely using two TM domains (15, 22). Recently, nsp6 was shown to contain six TM domains (22); however, the authors were unable to resolve which of two C-terminal hydrophobic domains can act as the final membrane-spanning region.Open in a separate windowFIG. 1.Schematic diagram of MHV RNA genome, indicating the proteolytic processing scheme of the replicase polyprotein and Western blot detection of MHV nsp6. (A) MHV-A59 linear RNA genome with the canonical representation of replicase, structural, and accessory genes. The replicase polyprotein intermediates and mature nsps generated during processing are depicted. The mature nsp6 replicase protein (hatched box) and the antibodies used to detect nsp6 and nsp8 (solid black boxes) are indicated. aa''s, amino acids. (B) Western blot analysis of nsp6. Whole-cell lysates were prepared from mock-infected (M) and MHV-infected (I) HeLa-MHVR cells, and the lysates were separated by 12.5% SDS-PAGE. Products were detected by probing with nsp6- or nsp8-specific antibodies.In this report, we show the first antibody-mediated detection of MHV-A59 nsp6 in virally infected cells. We also report the TM topology of nsp6, as determined by glycosylation tagging and N-linked glycosylation sequence insertion mutagenesis approaches, providing evidence that nsp6 contains six membrane-spanning segments with a large C-terminal tail exposed to the cytosol. Multiple alignment of the nsp6 amino acid sequences from each coronavirus group revealed a high level of conservation at the C-terminal end, suggesting an evolutionarily conserved function.To detect nsp6 replicase protein in MHV-A59-infected cells, we used a polyclonal rabbit antiserum directed against a peptide (PLGVYNYKISVQEL) from the C-terminal region of nsp6. We detected the replicase intermediate p150 (nsp4 to nsp11) and two nsp6-specific products of 23 and 25 kDa (Fig. ​(Fig.1B,1B, lane 2) in MHV-infected HeLa-MHVR (25) cells by Western blot analysis. We found similar mature products of nsp6 in MHV-infected murine cell lines 17Cl-1 and DBT (data not shown). The same MHV-infected cell lysate was used to detect nsp8 replicase protein with a specific antibody that also recognizes p150 (Fig. ​(Fig.1B,1B, lane 4). The reason for the existence of multiple forms of nsp6 is currently unknown, although posttranslational modification or alternative processing of nsp6 cannot be ruled out at this point. Future experiments will be directed at purification and analysis of the two forms of nsp6 detected here.To develop a framework for understanding the membrane topology of nsp6, we first performed nsp6 bioinformatics analysis. Five out of the seven bioinformatics tools predicted that nsp6 would encode seven TM domains, whereas two programs predicted that it would encode eight TM domains (Fig. ​(Fig.2).2). However, because both the N and C termini of nsp6 must be processed in the cytosol by the viral 3C-like protease (3CLpro), we expected nsp6 to encode an even number of TM domains and established a consensus TM domain prediction for nsp6 (Fig. ​(Fig.2,2, bottom row). The consensus provided a working model for generating plasmid DNA constructs for evaluating the membrane topology of MHV nsp6. First, we employed enhanced green fluorescent protein (EGFP) glycosylation tagging (EGFPglyc) experiments as previously used for determining the membrane topology of other viral replicase TM proteins (20, 22). This approach allowed us to determine the localization of the tagged protein based on the differences in the mobility of the endoglycosidase H (endo H)-treated protein versus that of the untreated protein by the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Based on the consensus topology model (Fig. ​(Fig.3A)3A) suggesting a maximum of eight TM domains, we generated a series of plasmid DNA constructs starting with the N-terminal putative TM1 domain, and successively larger constructs were fused at their carboxyl terminus in frame with EGFPglycV5. The plasmid DNAs were individually transfected into BsrT7 cells (6), and the newly synthesized fusion proteins were radiolabeled with 100 μCi of [³⁵S]methionine-cysteine per ml from 20 to 22 h posttransfection. Chimeric proteins expressed from the cell lysates were immunoprecipitated with V5 antibody, either endo H treated or mock treated, separated using 12.5% SDS-PAGE, and analyzed by autoradiography as described previously (15).Open in a separate windowFIG. 2.Summary of TM predictions for MHV nsp6 obtained from membrane topology bioinformatics tools. The nsp6 amino acid sequence (amino acids 3637 to 3923 in the MHV A59 genome are numbered 1 to 287 for simplicity) was analyzed for TM-spanning domains by the use of various bioinformatics tools, and the residue numbers with predicted TM domains are displayed. The consensus TM topology of MHV nsp6 used as a basis for the topology experiments is depicted at the bottom row (shaded in gray).Open in a separate windowFIG. 3.Determining the topology of nsp6 by the use of EGFPglyc and insertion of glycosylation consensus sites. (A) Schematics of a working topology model of MHV nsp6 (obtained from our consensus experiments) and nsp6-EGFPglycV5 fusion constructs generated for endo H assay. (B) Metabolic labeling and endo H treatment of nsp6-EGFPglycV5 fusion proteins. The nsp6-EGFPglycV5 fusion proteins expressed in transfected BsrT7 cells were radiolabeled from 20 to 22 h posttransfection, and then cell lysates were subjected to immunoprecipitation with V5 antibody, treated with endo H or left untreated, separated by 12.5% SDS-PAGE, and analyzed by autoradiography. (C) Map of plasmid DNA construct showing the sites of inserted glycosylation acceptor consensus sequences (NXS). The locations of glycosylation insertion in the nsp6-V5 construct are represented, with the amino acid number at the site of insertion. (D) Metabolic labeling and endo H analysis of glycosylation sequence insertion expression constructs of nsp6-V5. The plasmid DNAs (iNsp6-V5 constructs) were transfected and analyzed as described for panel B. (E) MHV-A59 nsp6 topology model, summarizing the results of EGFPglycV5 and glycosylation sequence insertion experiments. Amino acid positions indicated by the symbol “Y” were glycosylated and were positive by endo H assay, whereas those positions tested but found not glycosylated and negative by endo H assay are depicted by solid black horizontal lines. The inserted glycosylation acceptor sequence positions precede the letter i. Selected charged residues are shown in white characters on a black background. K, lysine residues; R, arginine residues; E, glutamic acid residues.We found that fusion protein products expressed from the reporter constructs (nsp6-35glycV5, nsp6-86glycV5, and nsp-165glycV5) were glycosylated, as shown by sensitivity to endo H treatment, indicating that the C-terminal end of these chimeric proteins must extend into the ER lumen (Fig. ​(Fig.3B,3B, lanes 4, 8, and 10). In contrast, the remaining reporter constructs were not sensitive to endo H treatment; therefore, the C-terminal end of the chimeric constructs must extend into the cytoplasm (Fig. ​(Fig.3B,3B, lanes 6, 12, 14, 16, and 18). Thus, these results indicate the presence of three luminal loops in nsp6. Identical results were obtained when we used PNGaseF (data not shown), which indicates that the lack of endo H sensitivity was not attributable to the protein transiting through the Golgi body, thereby rendering the protein insensitive to endo H treatment.To further investigate nsp6 topology in detail, we exploited a glycosylation sequence insertion mutagenesis approach (7) to create acceptor sequences in the region between amino acids 86 and 200 of nsp6 by the use of site-directed mutagenesis as described in reference 32 in order to independently investigate the topology, since bioinformatics predictions of the TM domains within this region differ (Fig. ​(Fig.2).2). Consensus glycosylation acceptor sites (NXS) were generated at four sites in the nsp6-V5 plasmid backbone by introducing single-codon insertions as depicted in Fig. ​Fig.3C.3C. All the glycosylation insertion constructs were expressed and analyzed by use of the endo H assay as described above. As expected, the parental nsp6-V5 protein is not glycosylated and did not show a mobility shift after endo H treatment (Fig. ​(Fig.3D,3D, lanes 1 and 2). In contrast, expression of 99iNsp6-V5 revealed evidence of endo H sensitivity (Fig. ​(Fig.3D,3D, lanes 3 and 4), indicating the ER luminal localization of the N99 introduced into MHV nsp6. This result is in agreement with those obtained with the nsp6-86glycV5 construct that is also endo H sensitive (Fig. ​(Fig.3B,3B, lanes 7 and 8). The insertion of glycosylation acceptor sequences at other sites yielded endo H-negative results (lanes 6, 8, and 10), indicating the possibility that the introduced NX(S/T) motifs (i) are localized in the cytosol, (ii) are localized within the membrane, or (iii) are not used, as the glycosylation site is not at least 12 amino acids away from the end of the preceding TM and 14 amino acids away from the beginning of the following TM (12 + 14 rule), thus rendering it inaccessible for glycosylation (1, 7, 30). Our results confirm and extend the results of a recent study (22) in which authors were unable to resolve whether TM6 or TM7 acted as the final TM domain. Our results indicate that TM6 is the final TM domain for MHV nsp6. We propose a topology model of MHV-A59 nsp6 in Fig. ​Fig.3E3E which is in accordance with the distribution of positively charged residues (positive inside rule; reviewed in reference 31), depicting the higher number of lysine and arginine residues facing the cytosolic side of the membrane and the majority of charged residues excluded from the TM domain. Taken together, the results presented above are consistent with a six-TM domain model of MHV nsp6. This report provides new information on the membrane topology of nsp6 and provides potential clues with respect to the assembly of the coronavirus replication complex.To determine whether the experimentally determined six-TM-spanning domain topology of MHV-A59 is conserved among coronaviruses, we performed MUSCLE (10) and ClustalW (29) multiple sequence alignment of nsp6 amino acid sequences representing group 1, group 2, and group 3 coronaviruses obtained from PATRIC (http://patric.vbi.vt.edu/) (27). The most striking observations were the amino acid sequence conservation in the C terminus of all nsp6 proteins and the conservation in the hydrophobicities within the putative TM domains (Fig. ​(Fig.4).4). This analysis revealed several conserved sites that may be important for the function of nsp6. We designated the conserved region between TM2 and TM3 the “KH loop” because of the invariant lysine and histidine residues that are present in the cytosolic loop (Fig. ​(Fig.3E),3E), although the function of these amino acids is not yet known. We also designated the hydrophobic region in the C-terminal tail the “conserved hydrophobic domain” (Fig. ​(Fig.4).4). We speculate that cysteine residue(s) within the region we designated the “conserved G(X)C(X)G motif” may be modified by palmitoylation, indicating that this region of nsp6 may have important functions in establishing protein-protein or protein-membrane interactions during the assembly of the viral replication complex. Additionally, for the MHV-A59 nsp6 protein, the NetPhosK 1.0 server (http://www.cbs.dtu.dk/services/NetPhosK/) predicted serine and tyrosine residues (serine 244 and tyrosine 250; see Fig. ​Fig.4)4) at the C-terminal region as sites of possible phosphorylation by epidermal growth factor receptor kinase and protein kinase C, respectively. Both predicted sites are highly conserved in all coronavirus nsp6 proteins (Fig. ​(Fig.4).4). Overall, our analysis revealed conserved features in the nsp6 C-terminal region whose importance in viral replication can be investigated using a coronavirus reverse genetics system.Open in a separate windowFIG. 4.Multiple sequence alignment (MSA) and percent sequence identity of coronavirus nsp6. The nsp6 amino acid sequences of 18 different coronaviruses were obtained from PATRIC (http://patric.vbi.vt.edu/) and aligned using MUSCLE and ClustalW software. The experimentally determined TM domains of MHV-A59 nsp6 were used as a reference for alignment. Unshaded boxes indicate the conserved TM domains that aligned with other coronavirus nsp6 sequences; the conserved hydrophobic domain (CHD) predicted by all the topology programs is indicated by gray shading. The residues of the peptide against which the nsp6 antibody was raised are boxed, with residue designations shown in boldface. Putative sites for palmitoylation (cysteine residue[s]) within the GXCXG motif) and phosphorylation (serine 244 and tyrosine 250 in MHV-A59 nsp6) are indicated. Percent identity (% ID) values are indicated. In MSA, the following notations were used: asterisk indicate invariant amino acids, colons indicate highly similar amino acids, and dots indicate similar amino acids. HCoV, human coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; BCoV, bovine coronavirus; BatSARS, bat severe acute respiratory syndrome coronavirus; BatCoV, bat coronavirus; SARSCoV, severe acute respiratory syndrome coronavirus; FIPV, feline infectious peritonitis virus; PRCoV, porcine respiratory coronavirus; TGEV, transmissible gastroenteritis virus; PEDV, porcine epidemic diarrhea virus; IBV, infectious bronchitis virus.     

Harmonised Australian Reference Intervals for Serum PINP and CTX in Adults

Bone turnover markers (BTMs) are classified as either formation or resorption markers. Their concentrations in blood or urine of adults are considered to reflect the rate of bone remodelling and may be of use in the management of patients with bone disease. Major inter-method differences exist for BTMs, and harmonisation of methods is currently being pursued at an international level. Based on published data, this article describes age- and sex-specific Australian consensus reference intervals for adults for serum procollagen type I amino-terminal propeptide (s-PINP) and serum β-isomerised carboxy-terminal cross-linking telopeptide of type I collagen (s-CTX).     

中国内蒙古缨齿藓属(紫萼藓科)1新变种——缨齿藓菱形变种

报道了内蒙古清水河黄土丘陵地区发现的紫萼藓科1新变种——缨齿藓菱形变种[Jaffueliobryum wrightii(Sull.)Thér.var.rhombicumX.L.BaiSarula],该变种与干旱山地岩面生境中的原变种缨齿藓[Jaffueliobryum wrightii(Sull.)Thér.]相似,生境的变化导致其形态发生变化,主要表现在上部细胞菱形和细胞壁背部强烈加厚,未分化的叶上部边缘细胞、中肋横切面细胞不分化,叶片长0.7~0.8mm,毛尖长0.8~1.3mm。文中对缨齿藓及其新变种的形态学特征,分布和生境进行了描述,并提供了显微照片,另外,列出了缨齿藓属5个种的检索表。     

长爪沙鼠发情周期规律与判定方法

目的研究长爪沙鼠发情周期,揭示发情规律,优化判定方法。方法连续18 d采集50只长爪沙鼠阴道上皮脱落细胞涂片,采用角化细胞计数法研究长爪沙鼠发情周期规律。比较瑞氏染色、HE染色和直接镜检判定发情周期4个时相的优缺点。结果长爪沙鼠的发情周期有稳定型、不稳定型、假孕三种类型。其中稳定型占68.6%,发情周期为(106.3±35.0)h,可分为4个时相。4个时相角化细胞的比例分别为发情前期(13.5±7.8)%、发情期(86.7±9.9)%、发情后期(27.9±12.8)%和发情间期(3.3±2.8)%。结论角化细胞计数能准确地判定长爪沙鼠的发情周期及各个时相。直接镜检法能快速反映阴道脱落细胞的形态。     

人TRAIL基因cDNA的克隆及其在COS—7细胞中的表达

ＴＲＡＩＬ(ＴＮＦｒｅｌａｔｅｄａｐｏｐｔｏｓｉｓｉｎｄｕｃｉｎｇｌｉｇａｎｄ)是最近克隆的肿瘤坏死因子(ＴＮＦ)家族的新成员,由于它的蛋白质结构和生物学效应类似于ＦＡＳ/ＡＰＯ1Ｌ,因此,也被称为ＡＰＯ2Ｌ。在低浓度下,ＴＲＡＩＬ能迅速地诱导多种肿瘤细胞系的?..