Construction of a new universal vector for insertional mutagenesis by homologous recombination

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neo^R) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neo^R are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neo^R and tk cassettes are separated by four unique cloning sites on the right-hand side of the neo^R cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

Antigen-specific,MHC-unrestricted T cells

The published record suggests that in the majority of cases the antigen is recognized by the T cell receptor (TCR) as a complex of a foreign antigen and amino acid residues contributed by the major histocompatibility complex (MHC) antigens, and the antigen-specific, MHC-restricted effector function is an unambiguous result of this process. Alternatively, the T cell receptor may recognize a particular conformational form of the antigen which is dictated by the allelic differences in the MHC, resulting also in MHC-restricted recognition. When, however, a T cell which phenotypically fulfills all the requirements necessary to perform antigen specific, MHC-restricted function, shows a lack of MHC restriction, there are two possible explanations: 1) In addition to the MHC-restricted, antigen-specific T cell receptor the cell expresses, or has newly acquired the expression of another, MHC-unrestricted (NK-like) receptor, or 2) The specific antigen recognized by the T cell receptor, is able to bind to the receptor and activate the T cell without being presented by the MHC molecule. While the first possibility has been extensively described in the literature as well as other articles in this issue, the second possibility has not been dealt with to the same extent and is the primary focus of this review.     

Distribution and immunolocalization of stachyose synthase in Cucumis melo L.

Indirect evidence for the site of stachyose biosynthesis has been provided by determining the occurrence and distribution of stachyose, raffinose and galactinol, the donor of the galactosyl moiety for stachyose synthesis, in Cucumis melo L. cv. Ranjadew. Studies of enzyme activities for the synthesis of these sugars and their distribution in different plant organs and isolates has led to the conclusion that stachyose is synthesized mainly in mature leaves and seeds. Nevertheless, stachyose-synthase activity varied with leaf age, the developmental stage of a plant, the growing season and the plant cultivar used. No stachyose or stachyose-synthase activity could be detected in isolated mesophyll protoplasts and chloroplasts, whereas both were found in a minor-vein-enriched fraction isolated from mature leaves. The conclusion that stachyose biosynthesis is associated with minor veins was confirmed by immunolocalization of the enzyme. Positive specific immunoreactivity of stachyose synthase with polyclonal anti-stachyose-synthase antibodies, labeled with protein A-gold, was detected in intermediary cells of leaf minor veins. The implication of this local synthesis of the main transport sugar for phloem loading in mature leaves of Cucumis melo is discussed.Abbreviation RUBPCase ribulose-1,5-bisphosphate carboxylase This work was supported by Deutsche Forschungsgemeinschaft. The excellent assistance of Ms. B. Müller in preparing the samples for electron microscopy is gratefully acknowledged. The authors thank Professor H.J. Schneider-Poetsch for anti-RuBPCase antibodies.     

Immunogold-EM localization of myrosinase in Brassicaceae

Summary Immunogold labelling has been used to study the cellular and subcellular localization of myrosinase (-thioglucosidase, EC 3.2.3.1), using LR-White acrylic resin and ultrahin sections from four different species of Brassicaceae;Brassica napus L.,Sinapis alba L.,Raphanus sativus L., and B.oleracea L. For immunolabelling, a polyclonal antibody raised in rabbit against a highly purified myrosinase fromSinapis alba was used. Western blots showed that the antiserum was specific against myrosinase from these species. Ultrathin sections were sequentially incubated with anti-myrosinase antiserum and with secondary antibodies conjugated with colloidal gold. Gold label was present in typical myrosin cells both in radicles and in cotyledons when observed in the electron microscope. The intracellular localization showed that myrosinase was present in myrosin grains in the myrosin cells in all four species of Brassicaceae.Abbreviations BSA bovine serum albumin - PBS phosphate buffered saline - PBS-T PBS with 0.5% v/v Tween-20     

The fine structure of the lateral eyes ofNeocarus texanus Chamberlin and Mulaik, 1942 (Opilioacarida,Acari, Arachnida,Chelicerata)

Summary Neocarus texanus, a primitive mite, bears two pairs of eyes, which are principally similar in ultrastructure. Each eye is covered externally by a cuticular cornea. It is underlain by flat sheath cells which send extensive processes into the retina. The retina is composed of distal and proximal cells. The 20 distal cells of the anterior eye are inversely orientated and form 10 disc-like rhabdoms. They represent typical retinula cells. Each rhabdom encloses the dendritic process of a neuron, the perikaryon of which is located outside the retina (proximal cells). The significance of this cell is not known. The retina is underlain by a crystalline tapetum. In the posterior eye 14 retinula cells form 7 rhabdoms in an arrangement similar to the anterior eye. The eyes of one side of the body are located within a capsule of pigment cells. Together the axons of the distal and proximal cells form the two optic nerves, one on each side of the body. The optic nerves leave the eyes anteriorly and terminate in two optic neuropils located in the brain.From structural evidence it is concluded, that the resolution of the eyes must be rather low.The peculiar proximal cells have not been observed previously in Acari. They probably resemble at best the eccentric cells and arhabdomeric cells of xiphosurans, scorpions, whip-scorpions and opilionids. Also, inverse retinae and tapeta of the present type have not been found in Acari until now, but are present in other Arachnida. Thus the eyes ofNeocarus texanus evidently represent a unique type within the Acari.     

Protein kinase C activating phorbolesters enhance the cyclic AMP response to parathyroid hormone,forskolin and choleratoxin in mouse calvarial bones and rat osteosarcoma cells

The protein kinase C-(PKC) activating phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 nmol/l) and phorbol 12, 13-dibutyrate (PDBU; 100 nmol/l) enhanced basal cyclin AMP accumulation in cultured neonatal mouse calvaria. The cyclic AMP response to parathyroid hormone (PTH; 10 nmol/l) and the adenylate cyclase activators forskolin (1–3 mol/l) and choleratoxin (0.1 mg/ml) was potentiated in a more than additive manner by TPA and PDBU. In contrast, phorbol 13-monoacetate (phorb-13; 100 nmol/l), a related compound but inactive on PKC, had no effect on basal or stimulated cyclic AMP accumulation. In the presence of indomethacin (1mol/l), TPA and PDBU had no effect on cyclic AMP accumulation in calvarial bones per se, but were still able to cause a significant enhancement of the response to PTH, forskolin and choleratoxin. PTH-, forskolin- and choleratoxin-stimulated cyclic AMP accumulation in rat osteosarcoma cells UMR 106-01 was synergistically potentiated by TPA and PDBU, but not by phorb.-13. These data indicate that PKC enhances cyclic AMP formation and that the level of interaction may be at, or distal to, adenylate cyclase.     

改变乙型肝炎病毒核心抗原基因3′端序列对其在原核细胞中表达的影响

乙型肝炎病毒(HBV)的核心抗原基因(C基因)编码185个氨基酸残基,在原核细胞或痘苗病毒系统中能表达并装配成27nm大小的核心抗原(HBcAg)多聚体颗粒。已证实HBV C基因3′端编码近40个氨基酸的碱基序列,不是表达形成HBcAg颗粒所必需的。用外源基因替换这部分序列,已表达出表面带有外源基因产物的杂合颗粒,它具有很好的免疫原性,成为新型的基因工程多决定簇颗粒载体疫苗。但我们的实验中发现,用另外的外源基因替换3′端序列能显著影响HBV C基因在大肠杆菌中的表达,不同组成的外源基因其影响程度有所不同。     

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)