Glucocorticoid receptors in lymphoma cells in culture: relationship to glucocorticoid killing activity

Mouse lymphoma cells in culture which are killed by adrenal steroids contain specific cortisol receptors that may be involved in the initial events of hormone action. The similarity of these receptors to those in hepatoma tissue culture cells, where adrenal steroids induce tyrosine aminotransferase, suggests that certain aspects of steroid action are similar in the two systems. In three steroid-resistant lymphoma cell populations specific binding was less than in the parent lines, suggesting that conversion to steroid resistance may be associated with changes in specific steroid binding.     

Evidence of estrogen receptors in normal human osteoblast-like cells

In seven strains of cultured normal human osteoblast-like cells, a mean of 1615 molecules of tritium-labeled 17 beta-estradiol per cell nucleus could be bound to specific nuclear sites. The nuclear binding of the labeled steroid was temperature-dependent, steroid-specific, saturable, and cell type-specific. These are characteristics of biologically active estrogen receptors. Pretreatment with 10 nanomolar estradiol in vitro increased the specific nuclear binding of progesterone in four of six cell strains, indicating an induction of functional progesterone receptors. RNA blot analysis demonstrated the presence of messenger RNA for the human estrogen receptor. The data suggest that estrogen acts directly on human bone cells through a classical estrogen receptor-mediated mechanism.     

汞盐诱导烟草悬浮细胞产生细胞编程死亡

【目的】阐明氯化汞处理烟草悬浮细胞产生的细胞死亡具有细胞编程死亡（programmed cell death,PCD）的特征和分子机制。【方法】采用形态和生物化学方法观察氯化汞处理细胞后的变化,并采用药理学方法,通过添加抑制剂和抗氧化剂阐明氯化汞引发的信号转导途径。【结果】氯化汞诱导产生的细胞死亡具有PCD特征,包括染色质浓缩、细胞核的TUNEL检测呈阳性和DNA ladder的形成。氯化汞诱导烟草悬浮细胞过氧化氢积累,过氧化氢酶和抗坏血酸等抗氧化剂能降低细胞死亡率。蛋白激酶抑制剂、磷脂酶C和磷脂酶D的抑制剂能显著地降低氯化汞引起的细胞死亡和过氧化氢积累。【结论】氯化汞引起的细胞死亡是一种PCD,蛋白激酶和磷脂信号介导的过氧化氢积累参与细胞死亡过程。     

RH 5849, a nonsteroidal ecdysone agonist: effects on a Drosophila cell line

The steroid molting hormone 20-hydroxyecdysone is the physiological inducer of molting and metamorphosis in insects. In ecdysone-sensitive Drosophila Kc cells, the insecticide RH 5849 (1,2-dibenzoyl-1-tert-butylhydrazine) mimics the action of 20-hydroxyecdysone by causing the formation of processes, an inhibition of cell proliferation, and induction of acetylcholinesterase. RH 5849 also competes with [3H]ponasterone A for high-affinity ecdysone receptor sites from Kc cell extracts. Resistant cell populations selected by growth in the continued presence of either RH 5849 or 20-hydroxyecdysone are insensitive to both compounds and exhibit a decreased titer of measurable ecdysone receptors. Although it is less potent than 20-hydroxyecdysone in both whole-cell and cell-free receptor assays, RH 5849 is the first nonsteroidal ecdysone agonist.     

氯化汞对草地贪夜蛾Sf9细胞及核型多角体病毒的遗传毒理研究

 【目的】试验以草地贪夜蛾细胞sf9作为受体，检测氯化汞对其生长发育的影响。【方法】采用台盼兰染料排斥法测定细胞活力，苏木素-伊红染色法检测细胞中的微核，对经氯化汞处理诱变的病毒AcMNPV 的DNA进行PCR扩增，产物经测序后进行分子突变分析。【结果】sf9细胞在4 ?g·ml-1的氯化汞浓度作用下，其细胞表面变得粗糙，分裂生长减慢，当剂量增大到7 ?g·ml-1时，便可观察到一些细胞的细胞膜破裂，在9 ?g·ml-1时，某些细胞的完整性受到破坏。用苏木素-伊红染色法检测细胞中的微核现象时，9 ?g·ml-1氯化汞处理区的微核率高达6.8%，有些细胞出现三核甚至多核的核裂现象，反映部分细胞的完整性受到一定程度的损伤。AcMNPV病毒经氯化汞短时间处理后接种于sf9细胞，多角体在sf9细胞中的形成数目较对照区少，异常多角体的比例增加，抽提经氯化汞处理的AcMNPV 的DNA，采取PCR技术进行扩增反应，对获得的扩增产物进行测序分析，发现DNA序列上的碱基有2处G→C、T→C的转换和碱基缺失的现象。【结论】一定剂量的氯化汞将会引起草地贪夜蛾sf9细胞及核型多角体病毒的损伤与致突变。     

Identification of nuclear receptors for VIP on a human colonic adenocarcinoma cell line

Vasoactive intestinal peptide (VIP) is a neuropeptide with broad tissue distribution. Although its precise function is unknown, it is thought to exert its effect, at least in part, by interacting with cell surface receptors. Nuclear receptors for VIP have now been identified by specific binding of 125I-labeled VIP to nuclei of a human colonic adenocarcinoma cell line (HT29) and by cross-linking of 125I-labeled VIP to its receptor on intact nuclei. In contrast, 125I-labeled transferrin shows only background binding to nuclei but significant binding to intact cells. Purity of the isolated nuclei was further substantiated by electron microscopy. The apparent molecular sizes of the VIP--cross-linked nuclear and cell surface receptors are similar but not identical.     

Presence of laminin receptors in Staphylococcus aureus

A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation. The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity. Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin. Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen. There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar. The molecular weight of the receptor was 50,000 and pI was 4.2. Eukaryotic laminin receptors were visualized by means of the binding of S. aureus in the presence of laminin. Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.     

Alloantigen recognition is preceded by nonspecific adhesion of cytotoxic T cells and target cells

T-cell receptors bind antigens only when the antigens are exposed on the cell surface. This can be studied best in the interaction of cytolytic T lymphocytes (CTL) with target cells because the recognition and binding event can be separated from the lytic phase. Studies with CTL clones specific for HLA-A2 and HLA-B7 demonstrated that conjugates of CTL's and target cells can be formed in the absence of specific antigen recognition. Furthermore, T-cell receptor and target antigen cannot interact unless there is conjugate formation. This indicates that nonspecific conjugate formation between CTL's and target cells precedes the recognition of specific antigen by the T-cell receptor.     

Self-nonself discrimination by T cells

The alpha beta T cell receptor (TCR) recognizes antigens that are presented by major histocompatibility complex (MHC)-encoded cell surface molecules by binding to both the antigen and the MHC molecules. Discrimination of self from nonself antigens and MHC molecules is achieved by negative and positive selection of T cells in the thymus: potentially harmful T cells with receptors that bind to self antigens plus self MHC molecules are deleted before they can mount immune responses. In contrast, the maturation of useful T cells with receptors that bind foreign antigens plus self MHC molecules requires the binding of their receptor to MHC molecules on thymic epithelium in the absence of foreign antigen. The binding of the TCR to either class I or class II MHC molecules directs differentiation of the selected cells into either CD4-8+ (killer) or CD4+8- (helper) T cells, respectively.     

Regulation of expression of the interleukin-2 receptor on hematopoietic cells by interleukin-3

Remarkable similarities in the intracellular and genetic events occur when lymphoid and hematopoietic cells are exposed to their specific growth factors. The interleukin-2 (IL-2) receptor, whose cell-surface expression is an absolute requirement for the growth and differentiation of lymphoid cells, was detected on various nonlymphoid hematopoietic cell types in this study. Cell lines consisting either of granulocyte-macrophage precursors or mast cells, which are dependent on interleukin-3 (IL-3) for their growth, expressed high levels of the IL-2 receptor on their surface. Analysis of the binding characteristics of these receptors with 125I-labeled recombinant IL-2 revealed that only receptors with low affinity for IL-2 were present on these cells. Addition of purified recombinant IL-3 to these cell lines led to an increase in IL-2 receptor gene expression within 1 hour in isolated nuclei. This IL-3--induced increase in the number of IL-2 receptors on the cell surface is maximal within 24 hours. Addition of 10,000 units of IL-2 to these cells had no apparent effect on their growth or differentiation. The presence of the receptor with only low affinity for IL-2 on hematopoietic cells and the regulation by IL-3 suggest that this receptor is involved in some important metabolic event in hematopoiesis.     

Regulated splicing of the Drosophila P transposable element third intron in vitro: somatic repression

In eukaryotic cells alternative splicing of messenger RNA precursors (pre-mRNA's) is a means of regulating gene expression. Although a number of the components that participate in regulating some alternative splicing events have been identified by molecular genetic procedures, the elucidation of the biochemical mechanisms governing alternative splicing requires in vitro reaction systems. The tissue specificity of P element transposition in Drosophila depends on the germline restriction of pre-mRNA splicing of the P element third intron (IVS3). Drosophila P element IVS3 pre-mRNA substrates were spliced accurately in vitro in heterologous human cell extracts but not in Drosophila somatic cell splicing extracts. Components in Drosophila somatic cell extracts that specifically inhibited IVS3 splicing in vitro were detected by a complementation assay. Biochemical assays for Drosophila RNA binding proteins were then used to detect a 97-kilodalton protein that interacts specifically with 5' exon sequences previously implicated in the control of IVS3 splicing in vivo. Inhibition of IVS3 splicing in vitro could be correlated with binding of the 97-kD protein to 5' exon sequences, suggesting that one aspect of IVS3 tissue-specific splicing involves somatic repression by specific RNA-protein interactions.     

Counting low-copy number proteins in a single cell

We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta2 adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells (Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.     

Loss of Responsiveness to Osmotic Stress in Maize Root： the Effect of Water Channel Blocker HgCl2

In order to investigate the effect of water channel blocker HgCl2 on the hydraulic resistance in roots of maize seedlings, a xylem pressure probe was used to monitor the changes in root xylem pressure in response to NaCl- or mannitol-induced osmotic stresses before and after the application of HgCl2. When the maize roots were subjected to 500 umol L-1 HgCl2 in root bathing solution, not only a considerable decline in xylem pressure （increase in xylem tension） was observed, but the loss of responsiveness of the plant to both salt- and mannitol-induced osmotic stresses in terms of xylem pressure change was seen as well when the transpiration rate of the plant was not significantly changed. The results are similar but different from the reversed osmosis by the Fenton reaction in the internodes of Chara coralline, showing that the mechanisms of water transport across cell membrane in plant roots are far more complicated than expected.     

Lectin-Induced Mucus Release in the Urn Cell Complex of the Marine Invertebrate Sipunculus nudus (Linnaeus)

The mucociliary urn cell complex of the marine coelomate Sipunculus nudus secretes mucus 4 to 5 minutes after being exposed to Lotus tetragonolobus and Ricinus communis I agglutinins. Surface binding of both lectins is confined to the secretory area of the urn cell complex and, like the release of mucus, is inhibited by the specific saccharides L-fucose and D-galactose or by incubation in L-fucosidase and D-galactosidase. Mucus secretion may therefore be initiated by the interaction of mucus-releasing stimuli with fucosyl or galactosyl residues of specific membrane receptors.     

Behavioral effects of progesterone associated with rapid modulation of oxytocin receptors

The ventromedial nuclei of the hypothalamus (VMN) are important for the control of feminine mating behavior, and hormone action within these nuclei has been causally related to behavior. Estradiol induces receptors for oxytocin in the VMN and in the area lateral to these nuclei over the course of 1 to 2 days, and progesterone causes, within 30 minutes of its application, a further increase in receptor binding and an expansion of the area covered by these receptors lateral to the VMN. The rapid progesterone effect appears to be a direct and specific effect of this steroid on the receptor or membrane, because it was produced in vitro as well as in vivo and was not mimicked by a variety of other steroids. The effect of progesterone occurred in the posterior part of the VMN, where oxytocin infusion facilitated feminine mating behavior; it did not take place in the anterior part of the VMN, where oxytocin infusion had no effect on mating behavior.