ITP患者骨髓及血液检测指标与疗效关系的探讨

目的：为观察血小板表面相关抗体Ｇ（ＰａＩｇＧ）、外周血血小板计数（ＢＰＣ）、平均血小板容积（ＭＰＶ）、骨髓巨核细胞计数（ＭＫ）与产板型巨核细胞比例（ＰＰＭＫ）对特发性血小板减少性紫癜（ｉｄｉｏｐａｔｈｉｃ ｔｈｒｏｍｂｏｃｙｔｏｐｅｎｉｃｐｕｒｐｕｒａ,ＩＴＰ）患者疗效的预计价值。方法：采用Ｃｏｕｌｔｅｒ全自动血球分析仪及双抗体夹心ＥＬＩＳＡ法等,对７３例ＩＴＰ患者治疗前和治疗后的ＢＰＣ、ＭＰＶ、     

Effects of probenecid and brilliant blue G on rat enteric glial cells following intestinal ischemia and reperfusion

The P2X7 receptor participates in several intracellular events and acts with the pannexin-1 channel. This study examined the effects of probenecid (PB) and brilliant blue G (BBG), which are antagonists of the pannexin-1 channel and P2X7 receptor, respectively, on rat ileum enteric glial cells after on ischemia and reperfusion. The ileal vessels were occluded for 45 min with nontraumatic vascular tweezers, and reperfusion was performed for periods of 24 h and 14 and 28 days. After ischemia (IR groups), the animals were treated with BBG (BG group) or PB (PB group). The double-labeling results demonstrated the following: the P2X7 receptor was present in enteric glial cells (S100β) and enteric neurons positive for HuC/D; enteric glial cells exhibited different phenotypes; some enteric glial cells were immunoreactive to only S100β or GFAP; and the pannexin-1 channel was present in enteric glial cells (GFAP). Density (in cells/cm²) analyses showed that the IR group exhibited a decrease in the number of cells immunoreactive for the P2X7 receptor, pannexin-1, and HuC/D and that treatment with BBG or PB resulted in the recovery of the numbers of these cells. The number of glial cells (S100β and GFAP) was higher in the IR group, and the treatments decreased the number of these cells to the normal value. However, the PB group did not exhibit recovery of S100β-positive glia. The cell profile area (μm²) of S100β-positive enteric glial cells decreased to the normal value after BBG treatment, whereas no recovery was observed in the PB group. The ileum contractile activity was decreased in the IR group and returned to baseline in the BG and PB groups. BBG and PB can effectively induce the recovery of neurons and glia cells and are thus potential therapeutic agents in the treatment of gastrointestinal tract diseases.     

陕西株庚型肝炎病毒(GBV-C/HGV )RNA NS3区 cDNA(4248nt～4465nt)的扩增、克隆和序列分析

目的　了解陕西株庚型肝炎病毒 (HGV,RNA/ GBV-C)的核苷酸序列特点 .方法　从陕西省西安市两位非甲、乙、丙、丁、戊型肝炎患者的血清中 ,应用反转录及套式 PCR技术 ,从血清中提取 RNA,经特异性的反转录引物 P4反转录成c DNA,以此为模板分别用 P3,P4及 P1 ,P2 两对引物进行PCR扩增 ,扩增到 2 18bp的 HGV RNA NS3区部分基因 ,将其克隆入 Pin Point TMXal- T载体 ,挑选阳性克隆并进行了序列分析 .结果　 SG2 与 HGV的核苷酸同源性分别为 85 .6 4%与84.48% ;与 GBV- C的核苷酸同源性为 86 .2 1%与 84.48% ,其二者之间同源性为 97.13% ;二者与 HGV的氨基酸同源性分别为 98.2 8%和 93.10 % ,与 GBV- C的氨基酸同源性也为98.2 8% ,93.10 % ,二者之间的氨基酸同源性达 94.83% .结论　庚型肝炎病毒 NS3区核苷酸同源性较高 ,同义突变率也较高 ,可能是一个对其生存环境较为适应的病毒 .     

心血管手术输血后庚型肝炎病毒感染与年龄的关系

目的 探讨心血管手术输血后庚型肝炎病毒（ＨＧＶ）感染与年龄的关系。方法 选取连续１６６例心血管手术患者进行前瞻性研究,按年龄分为Ａ,Ｂ两组,≥３６岁为Ａ组,〈３６岁为Ｂ组,测定手术前后血样标本的庚型肝炎病毒ＲＮＡ（ＨＧＶ ＲＮＡ）。结果 术后３个月ＨＧＶ总感染率４．２％。Ａ组较Ｂ组感染率高（Ｐ＝０．００６４）,体重大（Ｐ〈０．０００１）,输血量多（Ｐ〈０．０００１）,Ｂ组以先天性心脏病为主（Ｐ〈０．０００１）。结论 心血管手术输血后ＨＧＶ感染率为４．２％。小儿和青壮年感染率较中老年人低。提示心血管手术应早期施工。     

广东地区GBV-C/HGV5'NCR区序列变异的研究

目的 了解广东地区ＧＢＶ－Ｃ／ＨＧＶ５＇ＮＣＲ区的序列变异情况。方法 以ＧＢＶ－Ｃ和ＨＧＶ的５＇ＮＣＲ区安全同源的序列设计合成引物用于套式ＲＴ－ＰＣＲ。从广东地区的１０例ＧＢＶ－Ｃ／ＨＧＶ感染者血中扩增了１０个充列,分别克隆对ｐＵＣ１９、ｐＴ－Ａｄｖ载体。以３５Ｓ标记的双脱氧链末端终止法对这１０个序列分别进行正反两个方向的序列测定。结果 １０株充列相互间同源性最大为１００％,最小为８４．４％。与Ｇ     

Pharmacological characterization of type 1α metabotropic glutamate receptor-stimulated [35S]-GTPγS binding

1. The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [³⁵S]-guanosine 5′-[γ-thio]triphosphate ([³⁵S]-GTPγS) binding assay.
2. L-Glutamate increased the rate of [³⁵S]-GTPγS binding in a concentration-dependent manner (−logEC₅₀ (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [³⁵S]-GTPγS, 1 μM GDP, 10 mM MgCl₂, 100 mM NaCl and 100 μg membrane protein ml⁻¹. The L-glutamate (100 μM)-stimulated increase in [³⁵S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM).
3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K_D) for [³⁵S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (B_max) of 361±30 fmol mg⁻¹ protein.
4. Metabotropic glutamate receptor agonists, quisqualate (−logEC₅₀ (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [³⁵S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists.
5. (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [³⁵S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (K_D) of 34.0±7.8 μM to be calculated for this mGluR antagonist.
6. Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml⁻¹, 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [³⁵S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [³⁵S]-GTPγS binding by the residual PTX-insensitive G-protein population.
7. The modulation of [³⁵S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

     

Analysis of receptor-G protein interactions in permeabilized cells

Receptor-induced binding of the stable GTP analogue, guanosine 5-[-thio]triphosphate (GTP [S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus -toxin, binding of GTP [S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP [S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP [S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP [S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP [S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.Overall, this study indicates that receptor-stimulated GTP [S] binding to G proteins in permeabilized cells is a sensitive and rapid method for analyzing receptor-G protein interactions, which can be applied to a variety of cultured cells and for various receptor systems.     

Cell cycle checkpoints and DNA repair preserve the stability of the human genome

Summary Chemical carcinogenesis in the regenerating rat liver is cell-cycle-dependent. Proliferating hepatocytes were maximally susceptible to initiation by a single dose of benzo[a]pyrene diolepoxide I when at the G1/S border. Hepatocytes in early G1 or late S/G2/M were less susceptible and non-proliferating G0 hepatocytes were resistant to initiation. Radiation clastogenesis in proliferating human fibroblasts also is cell-cycle-dependent. Ultraviolet radiation (UV) induced maximal frequencies of chromosomal aberrations in synchronized cells that were at the G1/S border. Cells in early G1 or G2 were significantly less sensitive. For both initiation of chemical carcinogenesis and UV-clastogenesis, it appears that replication of damaged DNA is required and DNA repair before replication reduces cellular risk. If DNA repair is protective, cell cycle checkpoints which delay DNA replication and mitosis should augment this protective influence by providing more time for repair. The contribution of cell cycle checkpoint function to DNA repair during cell cycle-dependent clastogenesis was studied using ataxia telangiectasia (AT) fibroblasts. The AT cells displayed a defect in the coupling of DNA damage to checkpoints which control the G1/S and G2/M transitions and the rate of replicon initiation in S phase cells. UV-clastogenesis in AT cells was cell-cycle-dependent with irradiation at the G1/S boundary inducing 3-times more aberrations than treatment in G0 at the time of release into the cell cycle. Thus, DNA excision repair during the pre-replicative G1 phase was protective even in cells with defective checkpoint function. However, following irradiation at the G1/S border, AT cells displayed about 6-fold increased levels of UV-induced chromosome aberrations in comparison to normal human fibroblasts that were treated at this time. These observations indicate that secondary and tertiary DNA lesions that are produced during replication of UV-damaged DNA (replicative gaps and double-strand breaks) also depend on checkpoint function for repair. The replicon initiation and G2-delay checkpoints that operate after initiation of S phase appear to play a major role in protection against UV-clastogenesis.DNA is, in fact, so precious and so fragile that we now know that the cell has evolved a whole variety of repair mechanisms to protect its DNA from assaults by radiation, chemicals, and other hazards. This is exactly the sort of thing that the process of evolution by natural selection would lead us to expect. Francis Crick inWhat Mad Pursuit     

北京铁路部分人群散发性急性非甲非乙非丙型肝炎病原学分析

对北京铁路地区部分人群５８例散发性急性非甲非乙非丙型肝炎进行了ＨＥＶ和ＨＧＶ血清学调查。结果如下,抗－ＨＥＶ阳性２２例（３７．９３％）,抗－ＨＧＶ和ＨＧＶＲＮＡ阳性共１２例（２０．６８％）,其中３例为戊型和庚型肝炎病毒混合感染。戊肝、庚肝病毒感染均以３１～４０岁发病最多,且男多于女;戊肝主要发生在冬春季,多有肠道暴露史;庚型肝炎病毒感染无明显季节性,有肠道外暴露史。ＨＧＶ可单独感染,提示它可能有一定的致病性。     

The extracellular versus intracellular mechanisms of inhibition of TCR-triggered activation in thymocytes by adenosine under conditions of inhibited adenosine deaminase

The absence or low levels of adenosine deaminase (ADA) in humans result in severe combined immunodeficiency (SCID), which is characterized by hypoplastic thymus, T lymphocyte depletion and autoimmunity. Deficiency of ADA causes increased levels of both intracellular and extracellular adenosine, although only the intracellular lymphotoxicity of accumulated adenosine is considered in the pathogenesis of ADA SCID. It is shown that extracellular but not intracellular adenosine selectively inhibits TCR-triggered up-regulation of activation markers and apoptotic events in thymocytes under conditions of ADA deficiency. The effects of intracellular adenosine are dissociated from effects of extracellular adenosine in experiments using an adenosine transporter blocker. We found that prevention of toxicity of intracellular adenosine led to survival of TCR-cross-linked thymocytes in long-term (4 days) assays, but it was not sufficient for normal T cell differentiation under conditions of inhibited ADA. Surviving TCR-cross-linked thymocytes had a non-activated phenotype due to extracellular adenosine-mediated, TCR-antagonizing signaling. Taken together the data suggest that both intracellular toxicity and signaling by extracellular adenosine may contribute to pathogenesis of ADA SCID. Accordingly, extracellular adenosine may act on thymocytes, which survived intracellular toxicity of adenosine during ADA deficiency by counteracting TCR signaling. This, in turn, could lead to failure of positive and negative selection of thymocytes, and to additional elimination of thymocytes or autoimmunity of surviving T cells.