低氧预处理对大鼠海巴神经元缺氧耐受性和IL—1β表达 …

目的：观察低氧预处理对大鼠海巴神经元缺氧耐受性和白细胞介素－１β（ＩＬ－１β）表达的影响。方法：取培养１２ｄ的两组（对照组和低氧预处理组）培养神经元,同时置于缺氧环境（０．９Ｌ／ＬＮ２,０．１Ｌ／ＬＣＯ２）中培养２、４、８和１２ｈ。分别观察它们的形态变化和神经元存活数,并用抗ｒｈＩＬ－１β单克隆抗体进行免疫组织化学染色,观察缺氧对大鼠海马培养神经元ＩＬ－１β表达的影响。结果：经低氧预处理的海马神经     

Crystallization of purified recombinant human interleukin-1 beta

The gene for human interleukin-1 beta was cloned from SK-hep-1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL-1 beta) and as a variant with an additional sequence of three amino acids on the N-terminus (rIL-1 beta +). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast-performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on two-dimensional (2-D) gels as a single spot with a pI of 6.7 +/- 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL-1 beta have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P4(1) or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 A resolution. A structure determination based on these crystals is under way.     

Purification and characterization of human recombinant interleukin-1 beta

A human interleukin-1 (IL-1) beta cDNA was cloned, and the region coding for the mature protein was expressed in Escherichia coli. The 17-kDa biologically active product was purified in 40% yield to apparent homogeneity, without chaotropes, from the soluble fraction of sonicated cell lysates. The recombinant IL-1 beta was characterized by amino acid analysis, NH2- and COOH-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, and biological assay. Specific biological activity was 4.6 X 10(8) units/mg in a co-mitogenic IL-2 induction assay using cultured EL-4 T-lymphocytes. The molar extinction coefficient was determined to be 10,300 cm-1 M-1 at 280 nm. NH2-terminal sequence analysis revealed that 70% of the product begins with the Ala corresponding to the NH2 terminus of the natural protein, while 30% begins with the following Pro. No initiator Met was observed. Both of the sulfhydryl groups are reactive to Ellman's reagent and to iodoacetamide under nonreducing conditions, indicating that the Cys residues do not form disulfide bonds. S-Carboxamidomethyl-Cys-rIL-1 beta retained biological activity in the IL-2 induction assay. Circular dichroism suggested an extensive beta sheet structure for rIL-1 beta.     

Multiple charge isomers of human recombinant interleukin-1 beta

Human interleukin-1 beta (rhuIL-1 beta), obtained by DNA recombinant technology, was radiolabelled. Its isoelectric properties were determined by various analytical techniques such as high-voltage ultrathin layer isoelectric focusing (IEF) and chromatofocusing. The rhuIL-1 beta molecule had a molecular mass of 18 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. When examined by IEF on a polyacrylamide gel of 1 mm thickness in the pH range of 3.5-9.5, it was resolved into two broad bands appearing in the pH range of 6.2-5.8 and 5.5-5.2. Each of the two bands was further resolved into multiple bands when electrofocused on (i) a thinner gel of 0.5 mm thickness and (ii) a narrower pH range of 5-8. Upon chromatofocusing in a liquid column, it was possible to isolate various charged components of rhuIL-1 beta. However, all these components reacted to the antiserum to rhuIL-1 beta and displayed a molecular mass of 18 kDa suggesting the charge heterogeneity of rhuIL-1 beta.     

Inhibition of NMDA-induced outward currents by interleukin-1beta in hippocampal neurons

There is increasing evidence that a functional interaction exists between interleukin-1β (IL-1β) and N-methyl-d-aspartate (NMDA) receptors. The present study attempted to elucidate the effect of IL-1β on the NMDA-induced outward currents in mechanically dissociated hippocampal neurons using a perforated patch recording technique. IL-1β (30-100 ng/ml) inhibited the mean amplitude of the NMDA-induced outward currents that were mediated by charybdotoxin (ChTX)-sensitive Ca²⁺-activated K⁺ (K_Ca) channels. IL-1β (100 ng/ml) also significantly increased the mean ratio of the NMDA-induced inward current amplitudes measured at the end to the beginning of a 20-s application of NMDA. In hippocampal neurons from acute slice preparations, IL-1β significantly inhibited ChTX-sensitive K_Ca currents induced by a depolarizing voltage-step. IL-1 receptor antagonist antagonized effects of IL-1β. These results strongly suggest that IL-1β increases the neuronal excitability by inhibition of ChTX-sensitive K_Ca channels activated by Ca²⁺ influx through both NMDA receptors and voltage-gated Ca²⁺ channels.     

Inhibitory effect of human recombinant interleukin-1 alpha and beta on growth of human vascular endothelial cells

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen which stimulates the growth of endothelial cells. The mitogenic effect of ECGF was inhibited by addition of recombinant interleukin-1 (rIL-1) alpha or beta in a concentration dependent manner. The morphological change was not observed distinctly. In the condition without ECGF, both types of rIL-1 enhanced [3H]-thymidine uptake slightly, but failed to increase cell numbers. These data suggest the possibility that the effect of rIL-1 on EC is modulated by the presence of ECGF.     

低氧预处理对大鼠海马神经元缺氧耐受性和Jun基因表达的影响

目的 :观察低氧预处理对缺氧 复氧后大鼠海马神经元Jun表达的影响。方法 :取培养 12d的两组 (对照组和低氧预处理组 )神经元 ,同时置于缺氧环境 (0 .90L LN2 0 .10L LCO2 )中培养 4h后取出 ,置含 0 .10L LCO2 和空气的培养箱内复氧培养 2 4h和 72h ,于不同时间取出 ,观察神经元存活数 ,并用抗Jun抗血清进行免疫组织化学染色 ,观察Jun表达阳性和阴性神经元数目 ,计算Jun表达神经元所占百分率。结果 :经低氧预处理的海马神经元缺氧 复氧后Jun表达阳性神经元百分率较对照组明显减少 ,神经元存活数明显高于对照组。结论 :低氧预处理可使海马培养神经元对缺氧产生耐受 ,减少缺氧 复氧后神经元Jun的表达。提高神经元存活数     

Stimulation of human polymorphonuclear leukocytes by recombinant human interleukin-1 beta.

Leukocyte activation is a property of systemic infection. Animal experiments indicate interleukin-1 (IL-1) as a possible modulator, while contradictory results have been reported from in-vitro stimulation of isolated leukocytes. The purpose of the present study was to investigate the activation of isolated polymorphonuclear (PMN) leukocytes in vitro by preparations of recombinant human IL-1 beta and IL-1 receptor antagonist, which in earlier studies could elicit and abrogate, respectively, a sepsis-like syndrome in rabbits. They have also been shown to influence acute phase protein synthesis in mice and rats, and release of leukocyte cathepsin G in vivo. It was found that recombinant human IL-1 beta elicited a dose-dependent luminol-enhanced chemiluminescence response in isolated human PMN leukocytes in the dose range 8.8 x 10(-11)-8.8 x 10(-8) M. The effect could be blocked by prior treatment with the IL-1 receptor antagonist, indicating a direct effect on the specific IL-1 receptor. Preincubation by IL-1 beta enhanced the effect of a secondary challenge with phorbol 12-myristate 13-acetate or formyl-Met-Leu-Phe by 30-40%. The priming effect of rhIL-1 beta could also be blocked by the specific receptor antagonist. In this study, incubation of PMN leukocytes with rhIL-1 beta failed to induce degranulation of both azurophil (neutrophil proteinase 4/proteinase 3) and specific (lactoferrin) granules. rhIL-1 beta has been shown to induce degranulation in vivo, which is thus indicated as an indirect effect. We conclude that IL-1 beta is a direct and specific, but probably weak stimulator of the PMN leukocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of peripheral administration of recombinant human interleukin-1 beta on feeding behavior of the rat

This study was undertaken to investigate the changes in feeding behavior, including ambulatory activity, induced by a single injection of Interleukin-1 beta (IL-1) (2 micrograms/rat) at 18:00, just before the dark phase. For this purpose, we used the Gunma University-type automatic apparatus for continuous and direct measurement of ambulation and drinking. A significant decrease in food intake was observed for 12 hours after treatment with IL-1. Peripheral administration of IL-1 also produced a marked decrease in ambulatory activity within 3 hours which continued for 6 hours. In addition, IL-1 produced a marked decrease in drinking behavior during the first 6 hours. We reported here the changes in consummatory and ambulatory behavior of rats after acute administration of IL-1. The sickness which IL-1 produced may, at least in part, contribute to these phenomena, although precise mechanisms are still unknown.     

Crystallization and preliminary x-ray diffraction studies of recombinant human interleukin-1 beta

Recombinant human interleukin-1 beta has been crystallized into a tetragonal cell. The unit cell constants are a = b = 54.9 A, c = 76.8 A, and alpha = beta = gamma = 90 degrees. The crystals diffract to better than 1.9 A and are suitable for high resolution data collection. The crystallization conditions and general crystal data are presented.     

Constitutive expression of interleukin-1beta (IL-1beta) in rat oligodendrocytes

The RT-PCR analysis of RNA from progenitor and differentiated primary rat oligodendrocytes, and from the oligodendrocyte CG-4 cell line, shows the presence of the IL-1beta mRNA, the type I IL-1beta receptor and the IL-1 receptor accessory protein in these cells. In situ hybridization of a rat IL-1beta probe to primary progenitor and differentiated rat oligodendrocytes results in a positive signal. The double hybridization of the IL-1beta probe, together with an oligodendrocyte-specific differentiation marker, to sections of postnatal rat brain at different stages of differentiation is also positive. The double immuno-labelling technique utilized indicates coincidence of the signals on the brain slices. The results show that IL-1beta mRNA is constitutively expressed in rat brain oligodendrocytes from 1 day after birth onward. In agreement with this observation, CG-4 cells, primary progenitor and differentiated rat oligodendrocytes are positively stained by antibodies against IL-1beta. Postnatal brain slices from 1 and 4 day old and adult rats, labelled with a double immunofluorescence technique, are also stained by antibodies against IL-1beta. This signal coincides with that of antibodies against oligodendrocyte-specific surface markers. We conclude that IL-1beta is constitutively expressed in rat brain progenitor and differentiated oligodendrocytes.     

Amyloid beta proteins inhibit Cl(-)-ATPase activity in cultured rat hippocampal neurons

Cl(-)-ATPase in the CNS is a candidate for an outwardly directed neuronal Cl(-) transporter requiring phosphatidylinositol-4-phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid beta proteins (Abetas), on the Cl(-)-ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid beta proteins (1-40, 1-42 and 25-35) concentration-dependently (1-100 nM) and time-dependently (from 1 h to 6 day) decreased Cl(-)-ATPase activity and elevated intracellular Cl(-) concentrations ([Cl(-)]i), Abeta25-35 being the most potent. Addition of inositol or 8-Br-cyclic GMP completely reversed these Abeta-induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4-kinase, 10 microM wortmannin or 20 microM quercetin, but not by a PI 3-kinase inhibitor, 50 nM wortmannin or 10 microM LY294002. The PI, PIP and PIP2 levels of the plasma membrane-rich fraction were lower in the Abeta-treated cells as compared with each control. In the Abeta-exposed culture, but not in control, stimulation by 10 microM glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8-Br-cyclic GMP prevented the effect of Abeta-treatment on the neurotoxicity of glutamate. Thus, Abetas reduce neuronal Cl(-)-ATPase activity, resulting in an increase in [Cl(-)]i probably by lowering PI4P levels, and this may reflect a pre-apoptotic condition in early pathophysiological profiles of AD.     

Purification and characterization of the immunostimulatory properties of recombinant human interleukin-1 beta

In this study we have used a new method for human recombinant IL-1 beta (rIL-1 beta) purification and investigated its immunostimulatory biological activity. The IL-1 beta gene was cloned using a novel mRNA preparation from activated human blood monocytes. The purification protocol consists of extraction and two chromatographic steps using the new Soloza cation exchange resin. The purified protein was characterized electrophoretically, by amino acid analysis and reverse phase chromatography. The protein migrated on SDS-PAGE with a molecular weight of 18.200 but demonstrated the minor presence of aggregates (dimers and trimers). Specific activity of purified rIL-1 beta in comitogenic assay on mouse thymocytes was 10(8) U/mg protein. rIL-1 beta increased in a dose dependent manner proliferation of Con A-stimulated murine thymocytes, splenocytes, PHA-stimulated human peripheral blood lymphocytes and transformed B-cell lines. Comitogenic activity depended on the degree of lymphocyte preactivation and was similar to that of natural human IL-1 beta. rIL-1 beta enhanced IL-2 production by murine spleen cells and EL-4 cell line and IL-2 receptor expression by human peripheral blood mononuclear cells. It induced PGE2 release from human blood monocytes but had no effect on human neutrophil chemotaxis, phagocytosis and respiratory burst.     

Purification and characterization of recombinant human interleukin-1 beta produced in Escherichia coli

Recombinant human interleukin-1 beta (rIL-1 beta) produced in Escherichia coli was purified to homogeneity by a combination of mass ion exchange column chromatography, ion exchange and gel filtration high performance liquid chromatography. The purified rIL-1 beta had a molecular weight of 18 kD on SDS-polyacrylamide gel electrophoresis and an isoelectric point of 6.9 on analytical isoelectric focusing. These values were almost same as those of natural interleukin-1 beta. The amino acid composition and amino acid sequence of the amino terminal region were consistent with those deduced from the cDNA sequence. In addition, the primary structure was confirmed by peptide mapping with lysyl-endopeptidase on reverse phase HPLC. Besides rIL-1 beta with amino terminal Ala, two molecular species, [Met0] rIL-1 beta and [desAla1] rIL-1 beta, were also obtained. Biological and physicochemical properties of the three species of rIL-1 beta were compared.     

Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli

The high-level expression of human interleukin-1 beta in Escherichia coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion- and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N- and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1/LAF) assay. The specific activities determined with the IL-1/MCF and IL-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively.     

Biotinylation of reactive amino groups in native recombinant human interleukin-1 beta

Recombinant human interleukin-1 beta (rIL-1 beta) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94. Few additional singly modified rIL-1 beta products were obtained under these conditions, despite the presence of 15 lysine residues in this protein. These data support the view that the N terminus as well as the trilysine sequence (residues 92-94) are readily susceptible to chemical modification and are exposed on the surface of the protein. Chromatography of intact biotinylated rIL-1 beta by C4 reverse phase HPLC resolved a protein modified exclusively at the N-terminal alanine from two proteins modified singly at either lysine 93 or lysine 94. In addition, a protein product modified at lysine 103 was also obtained when rIL-1 beta was similarly modified with sulfo-NHS-biotin. Since the only difference between the two biotinylation reagents relates to spacer length and its associated hydrophobicity, these data suggest that lysine 103 is not as accessible to surface modification reagents as are lysine 93, lysine 94, or alanine 1. Initial experiments indicate that none of the modifications described above decrease thymocyte proliferation by more than one order of magnitude. Therefore, these amino acid residues are not crucial for bioactivity, and we anticipate the use of these monobiotinylated proteins in structure/function analysis of IL-1 beta.     

缺糖缺氧对体外培养海马神经元的影响

目的：观察缺糖缺氧诱导的培养海马神经元损伤。方法：取培养12d的海马神经元,在缺糖缺氧条件下分别培养0．5～4h后取出,换原神经元培养液在常氧条件下继续培养24h。用0．4％台盼蓝染色,检测神经元坏死,并用TUNEL法检测神经元凋亡,计算存活、坏死和凋亡神经元所占百分率。同时用图像分析仪测定存活、坏死和凋亡神经元的胞体面积、周长和等园直径。结果：培养的海马神经元急性缺糖缺氧后0．5～4h,随缺糖缺氧时间的延长,坏死神经元逐渐增多,缺糖缺氧后0．5～2h再恢复糖和氧供应后24h,凋亡神经元明显增多。图像分析的结果表明,坏死神经元的胞体面积、周长和等园直径均明显大于凋亡神经元。结论：缺糖缺氧可引起海马神经元严重损伤,在急性缺糖缺氧后0．5～4h引起的神经元死亡以坏死为多见,但在缺糖缺氧后0．5～2h再恢复糖和氧供应后24,神经元死亡则以凋亡为多见。