Increased expression of highly branchedN-linked oligosaccharides terminating inN-acetylglucosamine residues in neoplastic and sclerodermal chicken fibroblasts

Summary Although neoplastic cells often show a shift towards the expression of largerN-linked oligosaccharides compared to their normal counterparts, little consideration has been given to the possibility that these changes might be a more general phenomenon characteristic of certain neoplastic and non-neoplastic proliferative disorders. TerminalN-acetylglucosamine (GlcNAc) cluster antigen (TGCA) is an immunoreactive epitope(s) of highly branchedN-linked oligosaccharides terminating in GlcNAc residues. Here we have compared the expression of this antigen in normal, neoplastic and sclerodermal chicken fibroblasts by immunomorphological methods. TGCA was detectable in only a few, if any, fibroblasts of normal chicken skin or those cultured from chicken embryos. In contrast, the antigen appeared in 15 to 30% of chicken embryo fibroblasts transformed with avian sarcoma viruses and about 50% of neoplastic fibroblasts of both Rous sarcoma virus-induced fibrosarcomas and carcinogen-induced transplantable fibrosarcomas. Significantly, TGCA was also found in most activated fibroblasts in the skin of chickens with hereditary scleroderma. These results indicate that increased expression of highly branchedN-linked oligosaccharides terminating in GlcNAc residues is characteristic of both neoplastic and sclerodermal chicken fibroblasts. Investigation of this phenomenon may thus provide insight into biochemical pathways involved in neoplastic transformation and pathogenesis of a number of non-neoplastic proliferative connective tissue disorders such as scleroderma. Moreover, changes in the expression of TGCA-positive oligosaccharides (or their modified biochemical counterparts in mammalian species) may have considerable value for diagnosis of several connective tissue diseases.     

Increased collagen synthesis in myeloblastosis-associated virus-infected chicken embryo fibroblasts.

Chicken embryo fibroblasts infected with a non-transforming derivative of avian myeloblastosis virus [MAV-2(0)] showed a threefold increase in the biosynthesis of collagen compared to values in normal counterparts. In contrast, non-collagen protein synthesis was unchanged.     

鸡胚成纤维细胞cDNA表达文库的构建

鸡胚成纤维细胞(CEF)是研究鸡传染性法氏囊病病毒(IBDV)的主要细胞材料,而构建CEF的cDNA表达文库是筛选IBDV在CEF中的细胞受体,研究细胞嗜性的基础平台。采用Gateway技术构建CEF的表达文库,避免使用限制性内切酶切割cDNA,能够解决常规方法构建cDNA文库的技术缺陷。该技术将CEF的mRNA分离纯化后,以5′端生物素标记的Oligo(dT)primer为引物反转录后连接Adapter,层析柱纯化,通过BP重组反应构建cDNA入门文库,其平均滴度为1.1×106cfu/mL,文库总容量为1.2×107cfu,平均插入片段为2243bp,重组率为100%。通过LR重组反应将入门文库转换为表达文库,经测定平均滴度为5×105cfu/mL,文库总容量为5.5×106cfu,平均插入片段为2411bp,重组率为100%。结果表明,所构建的文库具有较高的重组率和较大的库容量,可作为较高质量的文库来研究IBDV的相关基因,为研究病毒受体和病毒入侵途径,进一步了解IBDV的致病机理奠定了基础。     

Enzymic synthesis of oligosaccharides terminating in the tumor-associated sialyl-Lewis-a determinant

The isomeric sialyl-Lea-terminating pentasaccharide derivatives, alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1 ----4)]-beta- D-GlcpNAc-(1----3)-beta-D-Galp-O(CH2)8COOMe and alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1 ----4)]- beta-D-GlcpNAc-(1----6)-beta-D-Galp-O(CH2)8COOMe, have been prepared by the action in sequence of a porcine submaxillary (2----3)-alpha-sialyltransferase and a human-milk (1----3/4)-alpha-fucosyltransferase on the chemically synthesized trisaccharides beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)- and -(1----6)-beta-D-Galp- O(CH2)8COOMe, respectively.     

Retinal ligatin recognizes glycoproteins bearing oligosaccharides terminating in phosphodiester-linked glucose

Ligatin is a filamentous, baseplate protein that binds and localizes peripheral glycoproteins to the external cell surface. Glycoproteins coisolated with ligatin from embryonic chicken neural retina and radiolabeled with ³²P are retained by an affinity column containing covalently bound retinal ligatin. Elution is achieved preferentially by α-glucose 1-phosphate and, to a limited extent, by mannose 6-phosphate. Treatment with endo-β-N-acetylglucosaminidase H prevents the proteins from binding to the column and results in the release of high-mannose-type oligosaccharides containing ³²P. The simplest of these oligosaccharides is unaffected by alkaline phosphatase unless the treatment is preceded by mild acid hydrolysis. Enzymatic and chemical analyses suggest that the phosphate is present in phosphodiester bonds linking penultimate mannose residues to terminal glucose residues.     

Independent expression of avian sarcoma virus in doubly infected chicken embryo fibroblasts.

Infection of a chicken cell with avian sarcoma virus requires division of the infected cell before synthesis of infectious progeny is initiated. This requirement for a cell division for the complete expression of avian sarcoma virus has been examined further with chicken embryo fibroblasts infected with two distinct viruses. Chicken cells infected with and producing a mutant of Rous sarcoma virus temperature sensitive for transformation (tsLA24PR-A) were arrested in G0 by depletion of serum factors from growth medium. These stationary cells continued to produce infectious progeny in the absence of further cell division. Superinfection of the stationary cells with the wild-type Prague strain of Rous sarcoma virus (PR-RSV-C) produced a stable double infection in these cells. Progeny of the superinfecting PR-RSV-C, however, were not detected until these cells underwent division after stimulation with fresh serum-containing medium. The addition of colchicine to these serum-stimulated cells, although not affecting production of the tsLA24PR-A, inhibited the appearance of progeny of the superinfecting PR-RSV-C. These experiments indicate that each avian sarcoma virus infection of a chicken embryo fibroblast requires division of the infected cell for production of that virus regardless of whether or not the cell is already producing a similar virus. The results suggest, therefore, that the requirement for a cell division represents a requirement for an event that controls virus expression in a "cis-acting" fashion specific for the provirus.     

Increased expression of highly sulfated keratan sulfate synthesized in malignant astrocytic tumors

Keratan sulfate (KS) proteoglycans are expressed on a subpopulation of microglia in normal adult brain. We previously showed the up-regulated expression of KS in one of glioblastoma cell lines using anti-KS antibody (5D4). However, it has not been clarified whether KS is expressed in brain tumors and is involved in their malignancy. In this study, 54 astrocytic tumors were investigated about KS-expression using Western-blot with 5D4. In six of 14 anaplastic astrocytomas (43%) and 23 of 34 glioblastomas (68%), KS was detected by 5D4. KS was hardly detected by 5D4 in diffuse astrocytoma, suggesting that KS-expression is significantly expressed in malignant astrocytic tumors. In immunohistochemistry, KS is highly expressed in cell surface of malignant astrocytic tumors. Taken together, KS might be associated with the malignancy of astrocytic tumors, and be useful for a prognostic factor of astrocytic tumors.     

Ubiquitin in stressed chicken embryo fibroblasts

Ubiquitin, a small 76-amino acid protein which is highly conserved in eukaryotic cells, occurs in several forms other than the free polypeptide. Among these are protein conjugates in which ubiquitin is covalently linked in lysylpeptide bond to lysl residues of other proteins and fusion proteins in which the amino-terminal domain is the precise ubiquitin sequence. Ubiquitin plays a role in cellular proteolytic degradation and in chromatin structure and has been postulated to be involved in the induction of a set of proteins which function during the cellular response to various kinds of environmental stress. We have measured the various forms of ubiquitin in cultures of chicken embryo fibroblasts under normal growth conditions and after treatment with a thermal or chemical stress. Levels of free ubiquitin fell slightly, ubiquitin conjugate levels rose shortly after stress began, and both then increased substantially as one of the cell's ubiquitin-encoding genes was activated by stress. The level of a protein synthesized as the carboxyl-terminal domain of one ubiquitin fusion protein was unchanged by a heat stress. The most dramatic effect was seen in the rapid disappearance of the ubiquitinated form of histone H2A, one of the major ubiquitin conjugates in cells in the interphase portion of their growth cycle. A significant rise in protein turnover was detected as a result of the stress, but occurred only when cells were removed from the stress condition. These results suggest that ubiquitin plays an important role both during and after stress, but fails to support hypotheses for ubiquitin and proteolysis in the activation of stress genes.     

Myosin synthesis in embryonic chicken fibroblasts

The rate of constitutive myosin synthesis was measured in cultures of replicating embryonic chicken skin fibroblasts by pulse labeling with [3H]leucine. These cells synthesized the 200,000-dalton heavy chain of myosin (MHC) at a rate of 3.2 x 10(3) molecules/cell/min. Additionally, an independent estimate of the MHC synthesis rate needed to maintain a constant level of constitutive MHC/cell was calculated from total protein content, percentage MHC, fibroblast doubling time, and MHC half-life. This calculated rate of approximately 2.9 x 10(3) molecules/cell/min was in close agreement with the measured rate. By comparison, the synthesis rate of myofibrillar MHC in fully activated muscle cell cultures was approximately 2.9 x 10(4) molecules/nucleus/min.     

A comparison of expression of neoplastic potential of carcinogen-transformed human fibroblasts in nude mice and in chick embryonic skin

Summary Human foreskin fibroblasts transformed by representative chemicals from five different classes of chemical carcinogens, some requiring enzymatic activation and direct acting carcinogens, produced cell populations that exhibited anchorage-independent growth and expression of neoplastic potential in either nude mice or chick-embryonic skin (CES). There is a high degree of correlation between tumor incidence and invasiveness of CES. The unique feature of CES is the rapidity of expression of cellular neoplasia and interpretation of the simulated tumor in 4 d as a simulated fibrosarcoma. This method represents a system that can be used to evaluate human carcinogens in vitro in 6 to 10 wk. This work was supported in part by AFSOR, F49620-80 and the National Cancer Institute, Bethesda, MD, R01-CA-25907.     

The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts

The addition of certain amino acid analogues (canavanine, hydroxynorvaline, o-methylthreonine) or a mild heat shock at 45 degrees C caused chicken embryo fibroblasts to increase rapidly the synthesis of three proteins (molecular weights 22,000, 76,000 and 95,000 daltons) to levels which dominate the cells biosynthetic capacity and exceed the level of synthesis of the major cell structural proteins. Actinomycin D blocked the increased synthesis of p22, p76 and p95 in both analogue and heat shock-treated cells, while cycloheximide addition during the "induction" period blocked formation of these proteins only in analoguetreated cells. The elevated levels of synthesis for this set of proteins began to decrease shortly after restoration of the normal amino acid or normal temperature, and the normal pattern of cell protein synthesis was found 8 hr later. Induction of a similar set of proteins was detected in mouse L cells and baby hamster kidney cells after treatment with amino acid analogues or heat shock. Several laboratories have reported synthesis of proteins with similar molecular weights in cells subjected to conditions that alter glucose metabolism, and we speculate that these proteins may be associated with a hexose transport system.     

Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

DNA repair and survival in UV-irradiated chicken embryo fibroblasts

The role of the pyrimidine dimer in cell killing, DNA synthesis and repair has been studied by utilizing the light-requiring DNA-repair mechanism of photo- reactivation in UV-irradiated chicken-embryo fibroblasts. Survival, as measured by colony-forming ability at 41°C, is increased in cells left in the light. The initial inhibition of DNA synthesis by UV is much less in light-treated cells, and levels reach that of unirradiated controls much faster than when the cells are left in the dark. The number of endonuclease-sensitive sites (dimers)_measured by an assay with a crude extract from M. luteus, rapidly decreases as the cells are allowed to photoreactive. However, in the dark, significant amounts of repair also occur, but at a much lower rate and with a lag phase of several hours. Unscheduled DNA synthesis occurs to a similarly low extent in both dark- and light-treated cells, confirming the finding that some amount of excision repair occurs that is light-independent. When survival is examined as a function of the number of dimers present, the dimers, not the non-dimer products, appear to be responsible for cell killing. In this study, the removal of dimers in vivo by photoreactivation has made it possible to demonstrate directly that dimers are primarily responsible for the deleterious effects of UV on DNA synthesis and survival.     

Direction of locomotion in clones of non-neoplastic fibroblasts and their neoplastic derivatives

The locomotion of cloned mouse fibroblasts, non-neoplastic and their spontaneously transformed neoplastic derivatives was compared by means of cinephotomicrography. The spontaneous transformants grow as invasive transplantable sarcomas, whereas the non-neoplastic fail to grow as tumors, and do not show the diagnostic characteristics of neoplastic cells in culture; these include certain morphologic alterations, growth in soft agar, and susceptibility to killing by activated macrophages. The non-neoplastic cells tended to maintain the same direction of locomotion in sequential 2.5 h periods, whereas the neoplastic cells did not. Thus, cells in all non-neoplastic lines exhibited a “persisten” walk while cells from the neoplastic lines had a random pattern of locomotion. No relationship between cell density and randomness of locomotion was observed, and the non-neoplastic cells appeared to grow as rapidly as the neoplastic cells. However, the neoplastic cells had higher rates of locomotion possibly associated with their invasive potential in vivo. The deficient amount of lamellar cytoplasm in the neoplastic cells and the high migration rate may account for their random pattern of locomotion.     

Type C viral gag gene expression in chicken embryo fibroblasts and avian sarcoma virus-transformed mammalian cells.

Sensitive radioimmunoassays were developed for avian type C viral gag gene-coded proteins. These assays were used to examine the restriction to virus production by avian embryo cells and mammalian cells transformed by avian sarcoma viruses. The results indicate that although a high-molecular-weight primary translational product of the gag gene is expressed, its cleavage and processing are incomplete. Furthermore, analysis of intermediate cleavage products provided information regarding the order of sequences coding for the individual viral proteins within the avian type C viral gag gene.     

Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish

Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3′ untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.