Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs

To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac alpha2-6Galbeta1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acalpha2-6Galbeta1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses.     

趋化因子受体CCR5反义RNA在真核细胞中的表达

目的 构建趋化因子受体CCR5反义RNA真核表达载体并获取重组假病毒颗粒以用于抗HIV-1研究，方法 用RT-PCR法从健康人外周血单个核细胞（PBMCs)中获得趋化因子受体CCR5翻译起始区的基因片段，并以正、反两个方面定向插入到真核表达载体pLXSN上，重组载体用脂质体转染剂（lipofectAMINE)转染PA317包装细胞，抗-G418克隆的细胞上清经逆转录后用荧光定量PCR（FQ-PCR）测定假病毒滴度，进一步感染NIH/3T3细胞。结果 CCR5正、反义RNA的真核表达载体。经PA317细胞包装形成的假病毒颗粒已成功地感染NIH/3T3细胞，目的基因在该细胞中得到整合与表达。结论 从PBMCs中获得的趋化因子受体CCR5基因片段通过逆转录病毒载体可转移至真核细胞中并得到表达，为进一步研究CCR5反义RNA的抗HIV-1作用奠定了基础。     

Reversible and irreversible inhibition of rat brain muscarinic receptors is related to different substitutions on bisquaternary pyridinium oximes

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [³H]-N-methyl-4-piperidyl benzilate ([³H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [³H]-4NMPB, with the highest potency (K_I=1.7–6 M) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (B_max) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.     

The cerebrospinal fluid transferrin/tau proteins. A study by two-dimensional polyacrylamide gel electrophoresis (2D) and agarose isoelectrofocusing (IEF) followed by double-antibody peroxidase labeling and avidin-biotin amplification

The cerebrospinal fluid (CSF) transferrin/Tau proteins were studied by two-dimensional polyacrylamide gel electrophoresis (2D) followed by immunoblotting and by agarose isoelectrofocusing (IEF), and subsequent double immunofixation, peroxidase staining and Avidin-Biotin Complex (ABC) amplification. The pattern of the Tau protein was similar but not equal to that of the transferrin (Tf). When a genetic variant of Tf was present in the serum, the same variant was also observed in the corresponding CSF Tf and in the Tau fraction. After neuraminidase treatment, both serum and CSF Tf moved to the Tau position on IEF and 2D. On 2D, no desialized precursors of the Tau proteins were detected, whereas the Tf precursors were always detected. No synthesis of the Tau globulin in the brain can, therefore, be inferred. In CSF not treated with neuraminidase, Tf is the only sialoglycoprotein clearly desialized, showing that the Tau fraction cannot be generated by neuraminidase action at CSF level. In fact, the treatment of serum and CSF proteins with neuraminidase produced a clear shift in the isoelectric mobility of all sialoglycoproteins. We clearly demonstrate that the Tau globulin is the result of neuraminidase activity not located in the CSF compartment. We suggest that Tf could be desialized by the action of neuraminidase at the brain level and then be "washed" into the CSF. Brain utilization of Tf, meeting the brain iron requirement, seems likely.     

Evidence for a displaceable non-specific [3H]neurotensin binding site in rat brain

Summary Levocabastine is a potent antihistamine drug, structurally unrelated to neurotensin. In rat and mouse brain but not in other animal species, it inhibited 60% of the [³H]neurotensin binding displaced by unlabelled neurotensin or neurotensin (8–13).The levocabastine-sensitive site or site 1 displayed high affinity properties for levocabastine (IC₅₀=25 nM) and was highly stereospecific (IC₅₀-value higher than 10 M for one of the isomers). Binding to the site 1 in rat brain corresponded to the [³H]neurotensin binding displaceable by 1 M levocabastine, whereas binding to the site 2 corresponded to the binding displaced by 1 M neurotensin when the site 1 was occluded by 1 M levocabastine.Both site 1 and site 2 appeared to be saturable. Scatchard plots obtained in rat bulbus olfactorius allowed to calculate a K _D-values of 7.1 nM and a B _max-values of 37.2 fmol/mg original tissue for site 1, while site 2 displayed a K _D-value of 0.7 nM and a B _max-value of 16.3 fmol/mg original tissue. The regional distributions of both sites showed marked differences. The site 1 was homogeneously distributed throughout all rat brain areas, whereas the amount of site 2 binding was markedly different in separate brain areas: bulbus olfactorius and substantia nigra had the highest amounts (8.9 and 7.8 fmol/mg tissue) while cerebellum had the lowest (0.4 fmol/mg tissue).In spite of its high affinity and stereospecificity, site 1 has to be considered as an acceptor or recognition site for [³H]neurotensin because of its species-link, low saturability and homogeneous distribution in all rat brain areas.On the other hand, site 2 had the characteristics of a physiological receptor: high affinity, saturability in the low nanomolar range and marked regional distribution in rat brain. Site 2 corresponds therefore most probably to the physiological neurotensin receptor. The foregoing experiments provide evidence for the presence of a drug displaceable, non-specific (=unrelated to a physiological receptor) neurotensin binding site in rat brain; levocabastine should be an important tool to occlude this site in order to reveal, by means of in vitro binding assays, the specific neurotensin binding site in rat brain.     

Effects of individual terphenyls and polychlorinated terphenyls on rat hepatic microsomal cytochrome P-450-dependent monooxygenases: structure-activity relationships

The effects of o-, m- and p-terphenyl, 2,4-dichloro-, 2,4,6-trichloro-, 2,3,5,6-tetrachloro-, 2,3,4,6-tetrachloro-, 2,4,4'",6- tetrachloro- and 2,3,4,5-tetrachloro-p-terphenyl, 2,3,4,5-tetrachloro-m- and o-terphenyl as inducers of hepatic drug-metabolizing enzymes were determined in immature male Wistar rats. o-Terphenyl, 2,4-dichloro-, 2,4,6-trichloro-p-terphenyl and 2,3,4,5-tetrachloro-o-terphenyl induced 4,4-dimethylamino antipyrine N-demethylase at total dose levels of 300 mol/kg and the 2,3,4,5-tetrachloro-p-terphenyl induced ethoxyresorufin O-deethylase (EROD). In contrast, none of the other terphenyls or polychlorinated terphenyls (PCTs) induced these enzyme activities. Previous studies have demonstrated that 2,3,4,5-tetrachloro-p-terphenyl did not exhibit a high affinity for the 2,3,7,8-tetrachlorodibenzo-p-trachlorodibenzo-p-dioxin (TCDD) receptor protein (EC₅₀= 6.6×10^–6M). In contrast, this study showed that 2,3,4,5-tetrachloro-p-terphenyl was more active than either 2,3,4,5-tetrachloro-o- or m-terphenyl as an inducer of EROD. Moreover, the competitive receptor binding EC₅₀ values for the latter two isomers were > 10^–5 M and this result was also consistent with their lack of EROD induction activity. Previous studies showed that analysis of the data for a series of 4-substituted-2,3,4,5-tetrachlorobiphenyls indicated that the p-terphenyl structural moiety (i.e. 4-substituent = phenyl) did not interact with high affinity with the receptor protein binding site. Since the 2,3,4,5-tetrachloro o- and m-terphenyls are also poor ligands for the receptor protein, this data and results from other studies indicate that PCT congeners (and commercial mixtures) are therefore unlikely to elicit significant 2,3,7,8-TCDD-like biologic or toxic effects in target species.     

Biochemical effects and drug levels in rats after long-term treatment with the specific 5-HT-uptake inhibitor,citalopram

The effects in rats of long-term administration of the potent, specific 5-HT uptake inhibitor citalopram have been investigated. Citalopram hydrobromide (MW=405) was given in the diet, 99 or 25 mol/kg daily, for 13 days or orally, 49 mol/kg twice a day, for 14 days. High plasma and brain levels of citalopram were found during the treatment period, whereas negligible amounts were found 24 h after withdrawal. The 5-HT uptake mechanism in blood platelets was completely blocked, since levels of whole blood 5-HT during and shortly (2 days) after treatment were decreased by 75–90%. The drug load after the two highest doses in terms of plasma drug levels was the same as in depressed patients treated with citalopram. Receptor binding technique ex vivo was applied to different brain parts to measure receptor parameters for several neurotransmitters. All data were evaluated by Eadie-Hoffstee analysis. No changes were seen in B _max and K _d for -receptors (³H-dihydroalprenolol) in frontal cortex, occipital+temporal cortex, whole cortex and limbic structures, 5-HT₂ receptors (³H-spiroperidol) in frontal and whole cortex, ₁-receptors (³H-prazosin) in rest of brain and DA D-2 receptors (³H-spiroperidol) in corpus striatum and limbic structures. The uptake mechanism for 5-HT as well as the inhibitory effect of citalopram on this uptake remained unaffected in brain synaptosomes derived from control and from citalopram (99 mol/kg)-treated rats. Thus long-term treatment with citalopram does not induce changes in neurotransmitter receptors as seen with most tricyclic as well as newer atypical antidepressants. Most striking is the lack of - and 5-HT₂ receptor down-regulation. Since citalopram clinically shows clear antidepressant activity, this down-regulation does not seem to be a prerequisite of antidepressant activity.     

测定白血病脑脊液中SIL—2R，IL—6表达的临床意义

为了探讨急性淋巴细胞性白血病 (ALL)患者脑脊液中可溶性白介素 - 2受体 (SIL - 2R)、白介素 6(IL - 6)的表达及意义 ,采用双抗夹心ELISA法测定 3 0例ALL患者脑脊液 (CSF)中SIL - 2R ,IL - 6水平 ,并与 10名正常者进行对照 .结果 :ALL合并中枢神经系统白血病 (CNS -L)组较CNS -L已缓解及未合并CNS -L组二者水平显著升高 (P <0 0 1) ;CNS -L已缓解组二者水平接近对照组 (P >0 0 5 ) ;骨髓缓解及好转组二者水平低于治疗无效组 .结果表明 :监测二者水平变化有助于CNS -L早期诊断、估计预后、判断疗效 .     

影响P2Z受体介导慢性淋巴细胞白血病细胞凋亡的各种因素及其机理研究

目的　探讨嘌呤能 P2 Z受体介导慢性淋巴细胞白血病 (CL L )细胞凋亡的影响因素及其机理。方法　在二价阳离子—— 1.0 mm ol/L Mg2 + 、Zn2 + 、Ca2 + 、Sr2 + 、Co2 + 、Ba2 + ,不同浓度的 EDTA或 EGTA,不同温度及在含 15 0 mm ol/L胆碱的介质中 ,将表达 P2 Z受体 [P2 Z(+) ]的 CL L细胞分别同 1.0 mm ol/L三磷酸腺苷 (ATP)或 0 .1m mol/L苯甲酰苯甲酸 ATP(Bz ATP)体外培养 8小时 ,以 DNA凝胶电泳、Td T法和流式细胞分析 (FCA )检测上述条件下细胞凋亡的诱导或抑制效应。结果　 Mg2 + 或 Ca2 + 能以剂量依赖性方式促进 ATP诱导 P2 Z(+)细胞凋亡 ,而 EDTA或 EGTA却以相反的方式抑制 P2 Z(+)细胞凋亡的发生 ;1.0 mm ol/L Zn2 + 可完全阻止 ATP诱导 P2 Z(+)细胞凋亡所产生的 DNA片段 ,但其它二价阳离子包括 1.0 mm ol/L Sr2 + 、Co2 + 、Ba2 + 却不影响 ATP的诱导 ;胆碱作为磷脂酶 D(PL D)的抑制剂 ,也可部分抑制 P2 Z(+)细胞凋亡产生的 DNA片段 ;当温度低于 10℃ ,可完全阻止 ATP诱导 P2 Z(+)细胞凋亡产生 DNA片段的发生。结论　 P2 Z受体介导 CL L细胞凋亡可能与核酸内切酶 ,PL D的参与密切相关。     

多西他赛+卡铂联合曲妥珠单抗方案对早期人表皮生长因子受体2阳性乳腺癌的新辅助治疗效果

目的探讨多西他赛+卡铂联合曲妥珠单抗(TCH)方案对早期人表皮生长因子受体2(HER2)阳性乳腺癌的新辅助治疗效果。方法回顾性分析2013年1月至2018年12月北京大学第一医院乳腺疾病中心经治的522例早期HER2阳性乳腺癌患者的临床资料,占同期收治早期浸润性乳腺癌患者的21.80%(522/2 394)。其中113例接受TCH方案进行新辅助治疗,年龄[M(QR)]52(13)岁(范围:23~69岁)。记录TCH方案新辅助治疗后病理完全缓解(pCR,ypT0N0M0期)的例数,采用Miller-Payne标准进行病理学评价。采用Kaplan-Meier法计算无病生存率和总体生存率,采用Log-rank检验比较组间生存差异。结果接受曲妥珠单抗规范治疗患者(294例)的无病生存率优于未规范治疗患者(177例)(84.4%比72.4%,χ²=4.095,P=0.046)。发生3~4级不良反应的患者占全部患者的15.9%(18/113),包括3~4级中性粒细胞减少12例,腹泻6例。31例患者获得pCR(ypT0N0M0),pCR率为27.4%(31/113)。pCR患者与非pCR患者的无病生存率和总体生存率无差异(91.8%比85.0%,92.5%比90.5%,P值均>0.05)。病理学评价为G4~5的患者无病生存率优于G1~3患者(89.6%比81.5%,χ²=5.340,P=0.021),而总体生存率的差异无统计学意义(91.4%比89.1%,χ²=1.008,P=0.315)。结论早期HER2阳性乳腺癌采用TCH方案行新辅助治疗的效果较好,新辅助治疗后病理学评价为G4~5的患者的无病生存率更高。