Ecotropic murine leukemia virus-induced fusion of murine cells.

Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [3H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells.     

Characterization and expression of murine PRELP

The cDNA sequence of the murine proline/arginine-rich end leucine-rich repeat protein (PRELP) gene was cloned by PCR-based techniques. The gene encodes a protein of 378 amino acids, which is four amino acid residues shorter than its human counterpart. This difference resides mainly in the amino terminal region of the mature protein, which is five amino acids shorter in the mouse than the human and has a lower arginine content. The remainder of the protein, including the structure of the leucine-rich repeats, the potential sites for N-linked glycosylation, and the disulfide-bonded domains are well conserved between species. In common with humans, the murine gene possesses three exons, with the translation initiation codon residing in exon 2 and the termination codon in exon 3. Exons 1 and 2 are separated by an intron of approximately 6.7 kbp, whereas exons 2 and 3 are separated by an intron of approximately 1.7 kbp. Western blot analysis of mouse cartilage extracts indicates that PRELP exists as a glycoprotein of approximately 55 kDa, as in human cartilage. Immunohistochemical and in situ hybridization analysis reveal that PRELP is expressed in cartilage throughout both fetal development and post-natal life, in contrast to the human where expression in cartilage is not apparent prior to birth. Northern blot analysis indicates that PRELP mRNA is also expressed in the developing embryo prior to skeletogenesis. The promoter region of the mouse PRELP gene possesses no TATA box in its proximal region, in common with humans, and shows differences in the conservation of elements known to be involved in regulating expression of the human PRELP gene.     

Identification and characterization of murine IRAK-M

Interleukin-1 receptor-associated-kinases (IRAKs) are signal transduction mediators of the Toll/IL-1 receptor family, which comprise several transmembrane proteins involved in host defense mechanisms. Today four different human IRAKs (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, hu-IRAK-4) and two murine IRAKs (mouse pelle like kinase (mPLK) and mu-IRAK-4) have been described. Here we report the identification and characterization of murine IRAK-M (mu-IRAK-M), a mouse homologue to human IRAK-M (hu-IRAK-M). These IRAK-M molecules show 71% sequence identity, a comparable cellular expression, and functional similarities with respect to signal transduction capacity and kinase activity, suggesting functional homology in signalling in human and mouse cells.     

Cloning of murine cysteine sulfinic acid decarboxylase and its mRNA expression in murine tissues

Cysteine sulfinic acid decarboxylase (CSD) is the rate-limiting enzyme for biosynthesis of taurine which is essential to biological processes such as development of the brain and eye, reproduction, osmoregulation as well as the anti-inflammatory activity of leukocytes. We report the cDNA sequence of murine CSD that predicts a polypeptide of 493 amino acids. This protein shares 98% and 90% of amino acids with rat and human CSD, respectively, indicating that it is a true ortholog of CSD. Northern blot analysis revealed that CSD mRNA is expressed in kidney and liver, and was not detected in lymphoid tissues and lung. The nucleotide sequence of murine CSD should be useful for genetic manipulation of the CSD gene.     

Altered placental morphology associated with murine trisomy 16 and murine trisomy 19

The morphology of placentas from trisomy 16 and trisomy 19 mouse conceptuses aged 12 to 18 gestational days was studied at the light microscopic level. Comparisons were made with placentas from normal littermate animals. Trisomy 16 placentas showed marked changes from normal: 1) the junctional zone showed little indication of normal morphologic differentiation throughout gestation; 2) clusters of germinal trophoblast cells persisted in the labyrinth throughout gestation, whereas these cells disappeared by gestational day 16 in the normal littermate placentas; 3) the labyrinth was reduced in size in the trisomic placentas, and the differentiation of the interhemal membranes was delayed. The size of the labyrinths from trisomy 19 placentas appeared to be decreased, but otherwise the placentas appeared to have normal morphology. These observations and others from the literature show that placental development is affected by the presence of a trisomic genome, and that different trisomies influence the development of the placenta differently. For trisomy 16, we propose that the striking changes of the junctional zone may be associated with the trisomy 16-related gene dosage effect for alpha- and beta-interferon cell surface receptors. Because of the homology for this and other genes on mouse chromosome 16 with genes on human chromosome 21, findings related to the altered development of the trisomy 16 mouse may be relevant to understanding some of the phenotypic variations associated with human trisomy 21, the Down syndrome.     

Effect of recombinant murine interferon-beta on the replication of murine cytomegalovirus

Pretreatment of mouse embryo fibroblasts (MEF) with recombinant murine interferon-beta (rMuIFN-beta) induced a high level of intracellular 2',5'-oligoadenylate (2-5A) synthetase activity. However, murine cytomegalovirus (MCMV) replicated under such condition, indicating that MCMV is relatively insensitive in vitro to rMuIFN-beta. Thus, there was a dissociation of 2-5A synthetase activity and antiviral activity against MCMV. In contrast to MCMV, the two parameters were closely associated in the case of vesicular stomatitis virus (VSV).     

Deoxyglucose transport of uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells

The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent K_m for deoxyglucose transport was about the same for all types of cells (1–2 mM). The V_max of secondary cultures of mouse embryo cells decreased from 6 nmoles/10⁶ cells/minute for sparse cultures to less than 1 nmole/10⁶ cells/minute for density-inhibited cultures. The V_max was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The V_max for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent K_i values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.     

Modulation of murine osteoarthritis

Up to nine out of 10 male STR/ORT mice develop osteoarthritis (OA) of the medial tibial cartilage at an early age. This has now been shown to be related to changes in the activity and distribution of monoamine oxidase which is related to the metabolism of catecholamines. Treatment with diclofenac sodium tended to normalize this activity but there was no significant histological improvement. It was therefore postulated that two influences were involved in the development of OA: a cellular and an extracellular factor. The first was improved by diclofenac sodium; the second, namely oedema of the matrix, was improved by tribenoside. In very preliminary studies, feeding the two drugs simultaneously resulted in 7/9 mice having no sign of OA.     

Identification and characterization of murine IRAK-2

Interleukin-1 receptor-associated kinases (IRAKs) are pivotal signaling elements of the Toll/IL-1 receptor (TIL) family, which play a role in innate immune responses by coordinating host defence mechanisms. Presently four different forms of human IRAK molecules are cloned (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, and hu-IRAK-4). In the murine system, only three genes have been identified so far, mouse Pelle-Like Kinase (mPLK), which corresponds to human IRAK-1, mu-IRAK-M, and mu-IRAK-4. Here we report the molecular cloning and characterization of murine IRAK-2 (mu-IRAK-2), a mouse homolog to human IRAK-2 (hu-IRAK-2). Murine and human IRAK-2 molecules show 67% sequence identity, they are ubiquitiously expressed, and both practically lack autophoshorylation kinase activity. The murine molecule reveals two remarkable differences to its human counterpart: it shows a C-terminal extension and it has no stimulatory effect on IL-1 induced NF-kappa B activation when compared to hu-IRAK-2, suggesting subtle functional differences in signaling by IRAK-2 in human and mouse cells.     

Establishment and characterization of murine macrophage hybrids

Proteose peptone-induced peritoneal macrophages from CBA/J (H-2k) mice have been fused to a hypoxanthine phosphoribosyltransferase-negative variant of the P388D1 (H-2d) murine macrophage cell line. Six hybrid clones were isolated following HAT selection and further characterized. Five of the six clones express class I antigens of both parental haplotypes by microelisa and by flow cytometric analysis. Class II antigen expression of both haplotypes was apparent following a 72-hr incubation of the hybrids with concanavalin A-stimulated rat spleen cell supernatant. However, I-Ad was expressed in all hybrids to a greater extent than I-Ak. Three clones with the highest level of I-Ak expression, E5, C2, and C4, were capable of antigen presentation to the I-Ak-restricted T-cell line, D10.G4.1. LPS induction of the hybrids resulted in a 2- to 15-fold increase in the amount of IL-1 produced relative to the P388D1 parent. Finally, in distinction to P388D1, all hybrids demonstrated increased Fc-mediated erythrophagocytosis of chromium-labeled antibody-coated erythrocytes. These murine macrophage hybrids appear stable and should serve as useful models in understanding the regulation of macrophage function.     

Purification and enzymological characterization of murine neurotrypsin

An increasing number of studies indicate that serine proteases play an important role in structural plasticity associated with learning and memory formation. Neurotrypsin is a multidomain serine protease located at the presynaptic terminal of neurons. It is thought to be crucial for cognitive brain functions. A deletion in the neurotrypsin gene causes severe mental retardation in humans. For a biochemical characterization, we produced murine neurotrypsin recombinantly in a eukaryotic expression system using myeloma cells. From the culture medium we purified neurotrypsin using heparin-, hydrophobic interaction- and immobilized metal affinity chromatography. For an enzymological characterization two fragments of agrin containing the natural cleavages sites of neurotrypsin were used as substrates. The highest catalytic activity of neurotrypsin was observed in the pH range between 7.0 and 8.5. Calcium ions were required for neurotrypsin activity and an ionic strength exceeding 500 mM decreased substrate cleavage. Site-specific mutations of the amino acids flanking the scissile bonds showed that cleavage is highly specific and requires a basic amino acid preceded by a glutamate residue on the N-terminal side of the scissile bond. This sequence requirement argues for a unique substrate binding pocket of neurotrypsin. This observation was further substantiated by the fact that almost all tested serine protease inhibitors except dichloroisocoumarin and PMSF did not affect neurotrypsin activity.     

Cloning and expression of murine lymphotoxin cDNA

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.     

Purification and characterization of murine protoporphyrinogen oxidase

The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4), has been purified to apparent homogeneity from mouse liver mitochondria. The purification involves solubilization from mitochondrial membranes with sodium cholate followed by ammonium sulfate fractionation and gel filtration on a Sepharose CL-6B column. The eluate is adjusted to 0.67 M (NH4)2SO4 and loaded onto a phenyl-Sepharose column. After salt washes, the enzyme is eluted with 0.5% sodium cholate and 0.5% Brij 35. The final step is high-pressure ion-exchange chromatography on a DEAE-5PW column. The purified protein has a molecular weight of approximately 65,000 by gel filtration chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band corresponding to a molecular weight of 65,000. The absorption spectrum of the purified enzyme shows no evidence of a chromophoric cofactor. Purified protoporphyrinogen oxidase has a Km for protoporphyrinogen IX of 5.6 microM with a Vmax of 2300 nmol mg-1 h-1. It utilizes meso- and hematoporphyrinogen at about 10% the level of protoporphyrinogen. The pH optimum is broad with a maximum at 7.1. There is no stimulation or inhibition by any tested divalent cations, and sulfhydryl reagents have no inhibitory effect on the purified enzyme.     

Enucleation and reconstruction of murine L-cell fibroblasts

Two types of teflon chambers are suggested for the procedures of enucleation and reconstruction of cells cultivated in the monolayer. The first chamber simplifies the enucleation and permits performing it on the standard cover glasses with a small volume of medium with cytochalasin B. The second chamber permits purifying the karyoplast fraction from intact cells by their adhesion on the cover glasses. Then the karyoplasts are sedimented by centrifugation on cytoplasts located on the cover glasses. This procedure promotes sticking of fragments and their subsequent fusion in the presence of polyethylene glycol. In total the number of reconstructed cells increases many times.     

Primary culture and phenotyping of murine chondrocytes

The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate into fibroblasts when they are subcultured, which is a major problem. This protocol, involving primary cultures to limit dedifferentiation, describes two different methods for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes, both of which represent hyaline cartilage) from mice. Mice are of particular interest for cellular and molecular studies, as many tools suitable for use in mice are available. In addition, rapid development of transgenic and gene-targeted mice provides powerful instruments for biological studies. The protocol can be divided into four stages: isolation of cartilage (15 min per animal), isolation of chondrocytes (2 h extended overnight), seeding of chondrocytes (1 h 30 min) and growth in culture (6 d). To obtain confluency of chondrocytes using this protocol takes 7 d. Methods for phenotyping chondrocytes are also provided.     

Cloning and characterization of murine EDF-1

Murine endothelial differentiation-related factor (mEDF-1) encodes a basic intracellular protein of 148 amino acids which is highly homologous to the human and rat polypeptides. mEDF-1 is expressed in most murine tissues tested and is evolutionary conserved. mEDF-1 expression is modulated in mouse development, since its expression is high early in development and decreases thereafter. Because EDF-1 has been isolated as a gene differentially expressed by exposure of endothelial cells to the Tat protein of HIV, we evaluated mEDF-1 expression in different cell lines derived from tumors which spontaneously develop in Tat transgenic mice. Cells isolated from adenocarcinomas and leiomyosarcomas express very high amounts of EDF-1, independently from their capability to secrete Tat. Tat transgenic mice also develop skin lesions which closely resemble human Kaposi's sarcoma. Since Kaposi spindle cells, which are the proliferative component of the sarcoma, differentiate from an endothelial precursor, it is noteworthy that spindle cells derived from Kaposi-like lesions of the Tat transgenic mice downregulate EDF-1 when compared to microvascular endothelial cells isolated from the same tissue.     

Cryopreservation of murine spermatozoa

Cryopreservation of mouse sperm provides an economic option for preserving the large number of mouse strains now being generated by transgenic and targeted mutation methodologies. The ability of a spermatozoan cell to survive cryobiological preservation depends on general biophysical constraints that apply to all cells, such as the avoidance or minimization of the formation of intracellular ice during cooling. This action is typically achieved by use of cryoprotectant substances and by controlled, slow rates of cooling. Superimposed on those general constraints may be special characteristics of mouse spermatozoa, such as more narrow, osmotically driven volume tolerance limits and the fact that relatively successful freezing can be obtained without the use of a permeating cryoprotective agent. The lack of important information regarding sperm cells fundamental cryobiological properties, including their osmotic and membrane permeability characteristics, has hindered progress in developing anything but empirically derived methods. Genetic differences between inbred mouse strains are reflected in motility and fertility characteristics of mouse sperm and contribute to the difficulty of developing successful cryopreservation methods. Recovery of live young from frozen sperm has been much more successful with sperm from hybrid mice than from most inbred strains. There have been no published reports of successful cryopreservation of rat sperm. Nevertheless, in mice, success in deriving live young from intracytoplasmic sperm injection using sperm frozen under suboptimal conditions raises the possibility of using this technique for the ultimate rescue of sperm regardless of the success of cryopreservation. This technique, however, requires additional development and verification of its efficacy before it will be suitable for general laboratory use. Although cryopreservation of mouse sperm is not yet universally successful, it can be used reliably to supplement cryopreservation of embryos and other germline cells or tissues for preserving biomedically important strains of mice for research.