GABAA receptor subunits in the rat hippocampus I: Immunocytochemical distribution of 13 subunits

The GABA_A receptor is a ligand-operated chloride channel. It has a pentameric structure. In mammalian brain different subunits are recruited from four gene subfamilies. Using immunocytochemistry, we investigated the distribution of the 13 GABA_A receptor subunits in the hippocampus of the rat. GABA_A receptor subunits were heterogeneously distributed within different hippocampal subfields. High concentrations of ₁-, ₂-, ₄-, β₃-, γ₂- and δ-immunoreactivities were observed within the molecular layer of the dentate gyrus, representing the dendritic area of the granule cells. In the hippocampus proper, the predominant GABA_A receptor subunits were ₁, ₂, ₅, β₃ and γ₂ that were located throughout the strata radiatum and oriens of CA1 to CA3. Immunocytochemical staining was there less prominent for ₄-, β₁-, β₂-, γ₃- and δ- subunits. In the hippocampus proper, the β₁ subunit was preferentially located in CA2. The ₄- and δ-subunits were somewhat more abundant in CA1 than in CA3. Numerous local circuit neurons in the hippocampus proper and the hilus of the dentate gyrus contained ₁-, β₂-, γ₂- and/or δ-subunits. ₃ and γ₁ were present only in minute amounts and no ₆-IR was detected in the hippocampal formation.

The distribution of the GABA_A receptor subunits indicates the existence of heterogenously constituted GABA_A receptor complexes within various hippocampal subfields, which may exert different physiological or pharmacological properties upon stimulation by GABA or its agonists.     

Impaired antigen receptor induced calcium mobilization in a phospholipase C-gamma1 deficient B cell line

B lymphocytes express phospholipase C-γ₁ (PLC-γ₁) and phospholipase C-γ₂ (PLC-γ₂) isozymes. However, the relative importance of these two isozymes in B cell signaling is not known. We report here the identification and analysis of a B cell line deficient in PLC-γ₁. Mature splenic B lymphocytes and a panel of cell lines representing pre-B, immature and mature B cell stages expressed phospholipase C-γ (PLC-γ), but not the β or δ isoforms of phospholipase C (PLC). While all the tested B cell lines and primary splenic B cells expressed PLC-γ₁ and PLC-γ₂ isozymes, the L1.2 B cell line exclusively expressed PLC-γ₂, but not PLC-γ₁ isozyme. The PLC-γ₁ deficient L1.2 B cells expressed levels of surface IgM comparable to that of PLC-γ₁ positive 70Z/3 B cell line. However, stimulation through the antigen receptor induced Ca⁺⁺ response in 70Z/3, but not L1.2 B cells, suggesting a requirement for PLC-γ₁ in antigen receptor induced Ca⁺⁺ signaling in these cells. The possible use for L1.2 cells in testing the regulation of PLC-γ₁ and PLC-γ₂ dependent calcium mobilization in B lymphocytes is discussed.     

Staphylococcal Protein A and Human IgG Subclasses and Allotypes

Staphylocoecal protein A binds molecules belonging to the IgGl, IgG2, and IgG4 sub-classes. IgG3 proteins generally do not bind, except for those coded by the two γ3 alleles, which are G3m(u⁻): G3m(b⁰,b³,b⁵,s,v) and G3m(b⁰,b³,b⁵,s,t,v). G3m(u) is located in the C_H2 domain. The difference between G3m(u⁻) and G3m(u⁺) IgG3 proteins correlates with the sequence at position 339 in the C_H2 domain—Ala and Thr respectively. There is another structural difference in the CH3 domain which correlates with protein A binding and non-binding: all IgG proteins that bind protein A have His at position 435, whereas those that do not, have Arg at that position.     

PI3K/Akt和JAK2/STAT3信号转导通路在SO_2抗大鼠肢体缺血再灌注致急性肺损伤中的作用

目的:探讨PI3K/Akt和JAK2/STAT3信号转导通路在二氧化硫(SO2)抗肢体缺血再灌注(I/R)致急性肺损伤中的作用。方法:应用双大腿根部绑扎止血带复制大鼠双后肢缺血再灌注肺损伤模型。在再灌注前20 min腹腔注射Na2SO3/Na HSO3;在再灌注前1 h静脉注射Stattic或LY294002。应用TUNEL、ELISA、Western blot等方法检测细胞凋亡、细胞因子表达及相关信号通路蛋白表达的情况。结果:与对照组相比,I/R组的MDA及MPO含量、肺系数、细胞凋亡指数、细胞因子表达以及p-STAT3、p-Akt蛋白的水平均显著增高;当应用Na2SO3/Na HSO3后,上述反映肺损伤的各项指标均下降。Western blot检测结果显示I/R后,肺组织中p-STAT3和p-Akt蛋白的水平均明显增加。而应用Na2SO3/Na HSO3后,p-Akt蛋白的水平继续增加,但p-STAT3蛋白的水平却减少(P0.05)。结论:JAK2/STAT3和PI3K/Akt信号通路都参与了SO2抗肢体缺血再灌注致急性肺损伤的作用。JAK2/STAT3通路的活化,能够使I/R损伤加重;相反,PI3K/Akt信号通路的活化,可以使I/R损伤减弱。此外,JAK2/STAT3和PI3K/Akt信号通路之间存在交互作用。     

IgG subclass distribution of anti-Rh,anti-Kell and anti-Duffy antibodies measured by sensitive haemagglutination assays.

The IgG subclass distribution of anti Rh antibodies (anti-D, ''anti-Du'', anti-c, anti-E), anti-Kell and anti-Duffy (anti-Fya) antibodies was measured by two haemagglutination techniques on microtitre plates. The first technique involved rabbit subclass specific antisera which were used to agglutinate red cells previously reacted with the patients'' antibodies at high concentration. The second, which was more sensitive, had an additional step by introducing sheep anti-rabbit antibodies (sandwich technique). By the sensitive sandwich technique we revealed, for anti-D antibodies: IgG1 8/19, IgG3 1/19, IgG1/IgG3 8/19, IgG1/IgG2/IgG3/IgG 41/19, IgG1/IgG4 1/19; for the Du reactive anti-D antibodies: IgG1 1/8, IgG1/IgG3 1/8, IgG1/IgG3/IgG4 6/8; for the anti-E antibodies: IgG1/IgG2/IgG4 2/3, IgG1/IgG2/IgG3/IgG4 1/3; for the anti-c antibodies: IgG1 2/5, IgG3 1/5, IgG1/IgG3 1/5; for the anti-Kell antibodies: IgG1 9/20, IgG1/IgG3 1/20, IgG1/IgG4 8/20, IgG1/IgG3/IgG4 2/20; and for anti-Duffy antibodies: IgG1 1/8, IgG1/IgG4 7/8. These results are partly at variance with previously published results.     

负载As2O3壳聚糖纳米粒的药代动力学及其毒性观察

背景：As₂O₃作为抗肿瘤药物具有不同程度的不良反应,目前尚没有可以较好降低As₂O₃不良反应的方法。 目的：观察负载As₂O₃壳聚糖纳米粒在体内是否有缓释作用,是否可以延长药物作用时间,减低As₂O₃毒副作用。 方法：将20只SD大鼠以抽签法随机分为As₂O₃组和壳聚糖纳米粒-As₂O₃组进行药代动力学测定,于给药前和给药后不同时间点测定血液中砷浓度;将40只SD大鼠随机分为壳聚糖纳米粒组、壳聚糖纳米粒- As₂O₃组、As₂O₃组、生理盐水组进行毒理学检测,检测血浆中谷丙转氨酶、谷草转氨酶、肌酸激酶、肌酐、尿素氮水平,并结合苏木精-伊红染色形态学观察心、肝、肾组织形态学变化。 结果与结论：壳聚糖纳米粒- As₂O₃组较As₂O₃组半衰期明显延长(P < 0.05)。除肌酸激酶外,As₂O₃组其他各指标均高于其他各组(P < 0.05);而含相同浓度As₂O₃的壳聚糖纳米粒As₂O₃组与壳聚糖纳米粒组、生理盐水组各项指标差异无显著性意义(P > 0.05)。As₂O₃组大鼠肝脏和肾脏均有较明显的病理学改变,壳聚糖纳米粒- As₂O₃组未见明显形态学改变。4组大鼠心脏均未见明显形态学改变。说明负载As₂O₃壳聚糖纳米粒在体内有缓释作用,可以延长药物作用时间,减轻As₂O₃的毒副作用。     

Oocytes from Xenopus laevis contain an intrinsic σ2-like binding site

In preparation for expression studies for rat brain σ-binding sites, Xenopus oocytes were tested for the presence of [³H]di-o-tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [³H]DTG-binding sites, a high-affinity site (K_d = 32 ± 6 nM, B_max of 45.7 ± 19 pmol/mg protein) and a low-affinity binding site (K_d = 1.3 ± 0.7 μM, B_max of 3.2 ± 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar K_d to that of the mammalian σ₂-binding site (32 vs. 38 nM). Comparison of the IC₅₀ values for inhibition of [³H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (−)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP), (+(-pentazocine and Zn²⁺, showed similarity in rank (r² = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [³H]DTG-binding site in oocytes represents a σ₂-like binding site.     

肺癌靶向通用载体hu3D3/cSA融合蛋白的制备和活性分析

目的 制备一种新型的肺癌靶向通用载体hu3D3与核心链霉亲和素(core-streptavidin,cSA)的融合蛋白hu3D3/cSA,并鉴定其活性.方法 PCR扩增cSA基因,克隆至质粒hu3D3/pET-22b(+)而获得表达载体hu3D3/cSA/pET-22b(+),并在E. coli BL21(DE3)中表达,镍亲和层析柱纯化hu3D3/cSA融合蛋白,采用TEA缓冲液进行复性.SDS-PAGE和Western blot鉴定融合蛋白四聚体的形成.FITC标记hu3D3/cSA,通过荧光显微镜分析hu3D3组分的抗原反应性和特异性,流式细胞术比较hu3D3/cSA和hu3D3与靶点的结合能力;ELISA和Western blot鉴定cSA组分结合生物素(biotin)的能力.结果 获得序列正确的hu3D3/cSA/pET-22b(+)重组子,融合基因在E.coli BL21(DE3)中主要以包涵体的形式表达.纯化复性后的融合蛋白能够形成四聚体复合物,保留了cSA结合生物素的活性,hu3D3组分能与肺癌细胞表面抗原结合,且结合能力与单体hu3D3相比有明显增强.结论 成功制备了具有结合靶抗原和生物素活性的hu3D3/cSA融合蛋白,同时改善了hu3D3组分结合其靶点的亲合力(avidity).因此,hu3D3/cSA可以作为一个通用的肺癌靶向载体,与多种生物素化的抗肿瘤药物或试剂联用,有望为多药物联合靶向治疗肺癌提供一种可行的方法.     

Characterisation of avian paramyxoviruses of serotype PMV-3 isolated from commercial turkeys in Great Britain

Avian paramyxoviruses isolated from turkeys in 1981 and 1982 were shown by haemagglutination inhibition tests and structural polypeptide analysis to be similar viruses of PMV-3 serotype but more closely related to PMV-3/turkey/Wisconsin/68 than to PMV-3/parakeet/Netherlands/ 449/75 or PMV-3/parakeet/England/l-83/82.     

HLA-DR antigens and phenotypes in Dutch coeliac children and their families

In a study on the HLA-DR antigens and phenotypes in a series of Dutch coeliac children and their first-degree relatives, the B-cell antigens of 36 unrelated coeliac children, 110 first-degree relatives of 33 of them, and 201 controls were typed with the two-colour fluorescence test. The most frequent antigen was HLA-DR3 (69%), followed by DR7 (36%). The distribution of DR phenotypes showed that the most frequent was DR3/other DR (25%), followed by DR3/DR7 (17%), DR3/DR4 (14%), and DR3/DR3 (14%). However, due to the frequency of certain antigens in the controls, only phenotypes DR3/DR3 (relative risk = 6.2), DR3/DR7 (relative risk = 6.4), and DR3/DR4 (relative risk = 6.2) were significantly associated with CD. The family study confirmed the segregation of the disease with phenotypes DR3/DR3 and DR3/DR7. The present results show that the association between CD and phenotypes DR3/DR3 and DR3/DR7 is not an exclusive characteristic of Southern coeliac children.     

生物相容性Fe3O4@SiO2纳米粒子的合成及性能

背景：Fe₃O₄纳米粒子具有良好的磁学特性,SiO₂具有良好的生物相容性,Fe₃O₄@SiO₂复合纳米粒子有望成为靶向治疗的载体。 目的：采用反相微乳液法合成生物相容性的Fe₃O₄@SiO₂纳米粒子。 方法：首先,以FeCl₃•6H₂O、FeCl₂•4H₂O、油酸、氨水等为原料,采用一壶化学共沉淀法合成油酸修饰的疏水性Fe₃O₄纳米粒子。随后,将油酸包裹的Fe₃O₄纳米粒子分散于环己烷中,然后将Triton-X100、正己醇及水在搅拌条件下加入到上述溶液,形成稳定的反相微乳液;在反相微乳液中,以氨水为催化剂,使正硅酸四乙酯水解、缩合,从而获得Fe₃O₄@SiO₂复合纳米粒子。 结果与结论：①透射电镜、X射线衍射显示：采用一壶化学沉淀法合成的Fe₃O₄具有尖晶石结构,平均粒径约为3.5 nm;微乳液法能将SiO₂成功包覆于Fe₃O₄表面,形成平均粒径为40 nm的均一Fe₃O₄@SiO₂复合纳米粒子。②磁性能分析显示：Fe₃O₄纳米粒子包裹后饱和磁化强度下降,但包裹前后矫顽力趋于零,均显示超顺磁性。③MTT结果显示纳米粒子与人脐静脉细胞融合细胞(EA.hy926)共培养24 h时Fe₃O₄@SiO₂组吸光度高于对照组(P < 0.05);细胞培养48,72 h,两组比较差异无显著性意义(P > 0.05)。结果表明经反相微乳液法合成的Fe₃O₄@SiO₂纳米粒子是一种优良的生物材料,其具有稳定、易分散及超顺磁性等特性。     

Differences in proliferation of the hematopoietic cell line TF-1 and cytokine production by peripheral blood leukocytes induced by 2 naturally occurring forms of human IL-3

BACKGROUND: A naturally occurring polymorphism in the coding region of the human IL3 gene leads to a change in amino acid residue 8 from proline to serine. OBJECTIVE: We sought to determine whether the 2 different forms of IL-3 varied in function. These different forms are available as recombinant proteins (recombinant human IL-3/proline 8 [rhIL-3/P8] and recombinant human IL-3/serine 8 [rhIL-3/S8]). METHODS: The erythroleukemic cell line TF-1 was incubated with varying concentrations of rhIL-3/P8 or rhIL-3/S8 to determine the capacity of each type of IL-3 to induce proliferation. Human leukocytes were primed with rhIL-3/P8 or rhIL-3/S8 for up to 24 hours and then stimulated with anti-IgE and assessed for leukotrienes (LTs), IL-4, and TNF-alpha. RESULTS: Proliferation of TF-1 cells was induced by both forms of IL-3 at 48 and 72 hours but to a greater degree by rhIL-3/P8. In contrast, the mean fold increase over control values of LT and IL-4 production was higher after priming the cells with rhIL-3/S8 versus rhIL-3/P8. Additionally, TNF-alpha production was greater (and reached significance only) for rhIL-3/S8. This activity was independent of IgE and thus directly stimulated by IL-3. Studies with basophil-enriched and basophil-depleted cell preparations revealed that LT production was evident only from the former and TNF-alpha only from the latter. CONCLUSION: We conclude that the 2 naturally occurring forms of human IL-3 have similar spectra of activities on cells with IL-3 receptors, but the 2 forms have reversed relative efficacies for promoting proliferation (rhIL-3/P8 > rhIL-3/S8) compared with priming or inducing mediator secretion (rhIL-3/S8 > rhIL-3/P8).     

四种靶向因子与柯萨奇病毒B3 VP1融合基因疫苗免疫效果的比较

目的:比较巨噬细胞源趋化因子(MDC)、补体片段C3d3、志贺毒素B亚单位(STxB)和小鼠β-防御素2(mBD2)分别与柯萨奇病毒B3(CVB3)VP1构建的融合基因疫苗及VP1基因疫苗对小鼠的免疫效果。方法:将雄性BALB/c小鼠随机分为6组,每组22只,分别于股四头肌注射pcDNA3、pcDNA3/VP1、pcDNA3/MDC-VP1、pcDNA3/VP1-C3d3、pcD-NA3/STxB-VP1和pcDNA3/mBD2-VP1,接种剂量为每次100μg/只,3周免疫1次,共3次。每次免疫后第14天内眦静脉取血,用微量中和试验滴定血清中和抗体滴度。第3次免疫后第21天,每组随机取3只小鼠,制备脾淋巴细胞,用CCK-8细胞计数法检测特异性CTL的杀伤活性;每组取3只小鼠以3LDLD50CVB3病毒攻击,第7天取血处死,检测血清病毒滴度;其余小鼠以5LDLD50的CVB3攻击,观察各组的生存情况。结果:除了pcDNA3对照组,其他各组的中和抗体滴度均随免疫次数的增加而提高(P0.01),第3次免疫后,pcD-NA3/MDC-VP1、pcDNA3/VP1-C3d3和pcDNA3/mBD2-VP1组的抗体滴度明显高于pcDNA3/VP1组(P0.01);pcDNA3/STxB-VP1和pcDNA3/mBD2-VP1组CTL杀伤活性显著高于其他各组(P0.01)。致死量病毒攻击后,pcDNA3/MDC-VP1、pcDNA3/VP1-C3d3和pcDNA3/mBD2-VP1组小鼠血中病毒滴度显著低于其他组(P0.01);pcDNA3/MDC-VP1和pcD-NA3/VP1-C3d3组小鼠生存率显著高于其他各组(P0.05)。结论:pcDNA3/MDC-VP1和pcDNA3/VP1-C3d3能诱导出较强的体液免疫和细胞免疫,能有效降低血中病毒滴度,获得较高的小鼠生存率;整体效果优于其他组。     

外源性H2S干预对O3致哮喘小鼠气道反应性和气道重塑的影响

 目的: 探讨外源性硫化氢(hydrogen sulfide,H₂S)对臭氧(ozone,O₃)致哮喘小鼠气道反应性和气道重塑的影响。方法: 将15只SPF级C57BL/6雌性小鼠随机分为对照组、O₃组和NaHS+O₃组,每组5只。O₃处理前0.5 h,对照组和O₃组腹腔注射生理盐水,NaHS+O₃组腹腔注射NaHS(H₂S的供体);O₃组和NaHS+O₃组隔天用O₃处理,对照组则呼吸清洁空气。持续4周后无创测定小鼠的气道反应性,随后进行气管环张力测定,通过肺组织HE染色测定支气管基底膜周径(Pbm)和管壁总面积(Wat)并标准化衡量气道重塑程度,并行AB-PAS染色测定肺组织中杯状上皮细胞的变化情况以进一步评价气道重塑程度。结果: 与对照组相比,O₃组小鼠的气道反应性显著增高;NaHS+O₃组明显低于O₃组,但与对照组相比差异无统计学显著性。与对照组相比,O₃组小鼠在乙酰甲胆碱刺激时的气管收缩力显著加大;NaHS+O₃组明显小于O₃组。与对照组相比,O₃组小鼠的支气管壁厚度明显加大;NaHS+O₃组显著小于O₃组但仍大于对照组。与对照组相比,O₃组小鼠肺组织中杯状上皮细胞占气道上皮细胞总面积的百分比显著上升;NaHS+O₃组显著低于O₃组但仍高于对照组。结论: 外源性H₂S对O₃所致哮喘的气道高反应性和气道重塑有缓解与保护作用,有望为临床药物治疗哮喘提供新靶点。     

Macrophage Priming and Activation During Fibrosarcoma Growth: Expression of c-myb, c-myc, c-fos, and c-fms

Macrophages (Mφ)³ function by a two-step process that includes priming (induction of cytokine and enzyme mRNA) and activation (production of effector molecules). The initial steps in Mφ priming involve the expression of certain proto-oncogenes that regulate expression of other genes. Because tumor growth primes Mφ to produce several suppressor monokines, we determined if cancer induced Mφ expression of these proto-oncogenes. Unstimulated peritoneal Mφ from tumor-bearing hosts (TBH) constitutively expressed the proto-oncogenes c-fms, c-fos, c-myc, and c-myb, whereas normal host (NH) Mφ had little or no expression of these proto-oncogenes. When Mφ were given a 24-h adherence priming stimulus, NH Mφ expressed c-fms and c-fos at levels equivalent to TBH Mφ constitutive expression. Adherence had little or no effect on c-fms and c-fos expression in TBH Mφ or on NH and TBH Mφ c-myc expression. c-myb expression was not induced in NH Mφ during adherence and was strongly decreased in TBH Mφ. Activation with a 1-h lipopolysaccharide-treatment increased NH and TBH Mφ expression of c-fms, c-fos, and c-myc, with higher expression of these proto-oncogenes in TBH Mφ. Activation failed to induce c-myb expression in NH Mφ and completely inhibited expression in TBH Mφ. Because c-fms, c-fos, and c-myc are normally expressed early during Mφ activation, our results suggest that tumor growth primes Mφ by inducing expression of these proto-oncogenes. c-myb is expressed in immature Mφ and is downregulated during Mφ activation. These observations explain why NH Mφ expression of c-myb was not induced and are consistent with reports that suggest TBH Mφ have not reached full developmental maturity. The induction of Mφ protooncogene expression during cancer may put Mφ in a primed state, which leads to earlier and stronger production of adverse suppressor and cytotoxic molecules.     

B细胞趋化因子对CVB3融合基因疫苗pcDNA3/C3d3-sVP1免疫效果的影响

目的:探讨B细胞趋化因子(B-lymphocyte chemoattractant,BLC)对柯萨奇病毒B3融合基因疫苗pcDNA3/C3d3-sVP1免疫效果的影响。方法:将BALB/c小鼠随机分为4组,分别肌肉注射pcDNA3、pcDNA3/BLC、pcDNA3/C3d3-sVP1和含pcDNA3/BLC与pcDNA3/C3d3-sVP1的混合质粒,于免疫后不同时间检测小鼠血清的中和抗体滴度、特异性CTL杀伤活性;以LD50的CVB3病毒液感染已免疫小鼠,检测小鼠血中病毒滴度,观察存活情况以评价各种疫苗的免疫保护作用。结果:小鼠的中和抗体滴度随免疫次数增加而提高,混合组中和抗体滴度(42.17±1.43)和特异性CTL杀伤率(41.3%±3.51%)均明显高于pcDNA3/C3d3-sVP1组(P0.05),而血中病毒滴度低于pcDNA3/C3d3-sVP1组;经致死量CVB3感染后,pcDNA3/BLC和pcDNA3/C3d3-sVP1混合免疫的小鼠生存率达44%,生存率高于其他各组。结论:BLC可显著提高C3d3-sVP1基因诱导的特异性免疫应答,增强基因疫苗对机体的免疫保护作用。     

Heterogeneity in terms of interleukin 4-dependent regulation of Fce receptor/CD23 expression on chronic B-lymphocytic leukemia cells

To discriminate the stages of maturation arrest of leukemic B cells, we have investigated the cell surface expression of FcεR_1l (H107 antigen) on leukemic B cells from 6 patients with chronic type B-lymphocytic leukemia(B-CLL) by a double staining method combined with cytoflorometry, and their production of soluble FceR_ll ⁺ by an ELISA technique. FceR_ll was expressed onμ⁺/δ⁻ cells of case 5 as well as on μ⁺/δ⁺cells of cases 1,2 and 4, but not on μ⁺/δ⁺cells in cases 3 and 6. The cultivation of leukemic cells with IL-4 not only increased the percentage of FceR_ll⁺cells but also enhanced the production of soluble Fcer_llin most cases. However, IL-4 had no effects on μ⁺/δ⁻/Fcεr_ll⁺ cells of cases 5, which appeared to correspond to a rather late