Specificity and mechanism of the histone methyltransferase Pr-Set7

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.     

Regulation of IL-2 beta receptor expression and beta-chain mRNA by human thymocytes.

The high affinity form of the human IL-2 receptor (IL-2R) has two known components, the IL-2R alpha (p55) and the IL-2R beta chain (p75). We have previously shown that recombinant IL-2 (rIL-2) could induce the expression of the alpha-chain (p55) on T cells and thymocytes, and increase this expression following suboptimal activation with concanavalin A (Con A) in combination with IL-2. An increase in the accumulation of IL-2R alpha-specific mRNA induced by rIL-2 in T cells and thymocytes had also been documented. We report here that the expression of IL-2R beta on the cell surface can be demonstrated on human thymocytes by the binding of Mik beta1, a MoAb directed against an epitope of the beta-chain. The IL-2R beta chain is constitutively expressed on freshly isolated thymocytes; this expression can be increased in thymocytes activated with Con A in combination with IL-2 or tetradecanoylphorbol 13-acetate (TPA). Blocking the formation of high affinity receptors with a MoAb directed against the alpha-chain of the receptor results in an increase in the display of IL-2R beta as evidenced by binding of MoAb Mik beta1. The accumulation of IL-2R-beta-specific mRNA is observed in freshly isolated thymocytes and it is increased in thymocytes cultured with rIL-2 alone, with Con A, and further enhanced by the addition of rIL-2 in combination with Con A or with TPA. Cyclosporine (CsA), which inhibits the accumulation of lymphokine-specific mRNA of thymocytes, does not inhibit the induction of the accumulation of IL-2R beta-specific mRNA. This is analogous to its effect on the expression of the alpha-chain (p55), and the accumulation of alpha-chain-specific mRNA.     

Expression of early genes in the rat brain after administration of corticoliberin into the neostriatum

In situ hybridization using oligonucleotide probes was used to study the effects of intrastriatal microinjection of corticoliberin on the expression of the early genes c-fos, jun B, c-jun, and NGFIA in the rat brain. Administration of corticoliberin (0.25 g) into the neostriatum induced the expression of mRNA encoded by the early genes c-fos, jun B, and NGFIA in both the neostriatum itself and in its efferent structures, particularly the nucleus accumbens and various parts of the cortex. Intrastriatal microinjection of corticoliberin had no effect on the expression of mRNA for the oncogene c-jun in the brain. These results suggest that neuronal activation in the neostriatum and its projection targets manifest as the expression of early genes is one of the mechanisms underlying the adaptive effects of corticoliberin in stress.     

Signalling via CD28 of human naive neonatal T lymphocytes.

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the 'naive' neonatal T cell response to anti-CD2-mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti-CD2 antibodies in the presence or absence of anti-CD28 MoAb. With anti-CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL-2, whereas adult T cells proliferated vigorously, with significant IL-2 production. Costimulation with anti-CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL-2 secretion was increased in the presence of anti-CD28 MoAb, neonatal T cell IL-2 production remained lower than in adults. In contrast, enhancement of IL-2 mRNA expression in neonates was similar to adult levels. Anti-CD28 MoAb costimulation increased NF kappa B levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti-CD2 may therefore be due to decreased NF kappa B induction, reduced IL-2 mRNA expression and deficient IL-2 production. Although anti-CD28 MoAb costimulation enhances all of the above signals, NF kappa B and IL-2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate.     

Mouse monoclonal antibodies to the human C3b receptor

Mouse monoclonal antibodies were raised against the human C3b receptor (CR1) molecule that had been purified from solubilized erythrocytes membranes. Four hybridomas were selected, cloned and expanded because their supernatants reacted strongly with insolubilized CR1 by ELISA and intensely stained B-dependent areas of the spleen and glomerular podocytes by indirect immunofluorescence. The four monoclonal antibodies, named J3D3, J8B10, J3B11 and J7C2, were IgG1 immunoglobulins. J3D3 immunoprecipitated two protein bands of apparent mol. wts 200,000 and 220,000 from 125I-surface-labeled human erythrocytes, which correspond to the two major allotypic forms of CR1. By indirect immunofluorescence, monoclonal antibodies stained polymorphonuclear leucocytes (PMN), most peripheral blood B-cells and a small subset of peripheral blood T-cells. J3D3 bound to CR1 on erythrocytes, PMN and lymphocytes with an affinity of 1-3 X 10(9) M-1 and recognized 170-1330 antigenic CR1 sites with an average of 740 sites/erythrocyte in 100 healthy individuals, approx. 50,000 sites/PMN and 15,000 sites/lymphocyte. There was a bimodal distribution of CR1 numbers on erythrocyte in the normal population. The four monoclonal antibodies similarly inhibited CR1-mediated decay of preformed cell-bound alternative- and classical-pathway C3 convertase sites. Two antibodies, J3D3 and J3B11, inhibited C3b-dependent rosette formation with lymphocytes, although much less efficiently than F(ab')2 polyclonal anti-CR1 antibody. Differences that were observed in the relative capacity of the antibodies to inhibit some of the functions of CR1 and in their ability to compete for binding of 125I-J3D3 to CR1 on erythrocytes, suggested that they are directed against different epitopes on CR1. Monoclonal antibodies provide useful means to assess and analyze the biological and immunoregulatory functions of the C3b receptor.     

人参三醇皂甙对人淋巴结细胞IL—5基因表达的促进效应

应用mRNA麦胚无细胞体外转译体系,动态地观察了人参三醇皂甙(PTGS)对人淋巴结细胞IL-5基因表达的促进效应。结果表明,PTGS可以明显促进PHA活化人淋巴结细胞分泌IL-5,最大促进效应可达66.67％。PTGS+PHA共刺激后人淋巴结细胞浆IL-5 mRNA转译IL-5的量明显高于单纯PHA组,最大促进效应40％。上述结果首次证明PTGS对IL-5的促诱生效应是通过调节IL-5基因表达而实现的。     

组成型一氧化氮合酶的表达与胃癌的侵袭转移和预后的关系

目的　观察胃癌组织中cNOSmRNA表达分布情况 ,探讨其与胃癌侵袭转移和预后的关系。　方法　运用原位杂交技术 ,检测 119例原发性胃癌标本中cNOSmRNA的表达 ,并对所获得的 82份病例随访资料进行生存分析。　结果　胃癌组织中胃腺癌细胞、血管 淋巴管内皮细胞及巨噬细胞中均可见有cNOSmRNA表达 ;肿瘤细胞分化程度越低、恶性度越高 ,胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA阳性表达率就越高 ;胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA阳性表达率与胃癌侵袭深度、淋巴结转移以及TNM分期呈正相关 ;胃癌血管 淋巴管内皮细胞cNOSmRNA阳性表达者术后生存率较低 ;巨噬细胞中cNOSmRNA表达与胃癌组织学分型、侵袭深度、淋巴结转移、TNM分期及患者术后生存期无关。　结论　胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA表达情况与肿瘤恶性程度、侵袭和转移潜能有关 ,血管 淋巴管内皮细胞上cNOSmRNA阳性表达提示患者预后不良     

Antigen-Induced Activation of Hypothalamic Cells (assessed by expression of the c-fos gene)

The aim of the present work was to study the activation of the expression of the c-fos gene (by in situ hybridization) in cells from rat (Sprague Dawley) hypothalamic structures 0.5, 2, 6, and 16 h after i.v. injections of tetanus toxoid (200 g/kg). Tetanus toxoid was selected as the antigen because it does not induce any general non-specific body reactions. Control animals received i.v. doses of apyrogenic physiological saline. The number of c-fos mRNA-positive cells in all the hypothalamic structures studied was insignificant 30 min after injections of tetanus toxoid. c-fos mRNA-positive cells were seen in the posterior, lateral, and anterior hypothalamic fields and in the dorsomedial and ventromedial hypothalamic nuclei 2 h after injections of tetanus toxoid. The intensity of c-fos mRNA expression decreased in the posterior, lateral, and anterior hypothalamic fields 6 h after injections of tetanus toxoid. The maximum number of c-fos mRNA-positive cells in the anterior field and the paraventricular nucleus of the hypothalamic induced by tetanus toxoid, as compared with reactions to administration of physiological saline, were seen at 6 h. Administration of tetanus toxoid and physiological saline did not active the synthesis of c-fos mRNA in the arcuate or supraoptic nuclei at any time point. The number of c-fos mRNA-positive cells returned to baseline by 16 h after tetanus toxoid injections. Thus, this study revealed the temporospatial pattern of activation of hypothalamic structures in response to exposure to an antigen.