His-tag不影响RSV重组蛋白G1F/M2的免疫原性

目的：观察His-tag是否影响RSV重组蛋白G1F/M2的免疫原性。方法：PCR扩增G1和F/M2基因片段,插入表达载体pET-His和pET-DsbA-His中,转化E.coli BL21（DE3）,IPTG诱导表达,采用Ni^＋螯合亲和层析法纯化得His-G1F/M2和DsbA-His-G1F/M2,将后者用凝血酶消化,再经Ni＋螯合亲和层析法纯化得G1F/M2,将His-G1F/M2和G1F/M2免疫BALB/c小鼠,用ELISA测定抗体滴度,MTT法测定细胞毒性T细胞活性（CTL）。结果：两种蛋白在BALB/c小鼠中诱导的RSV特异性抗体和CTL活性无显著差异。结论：His-tag不影响RSV重组蛋白G1F/M2的免疫原性。     

125I标记抗人CD40 mAb 5H6在荷人卵巢癌(HO8910)BALB/c裸鼠体内分布及放射免疫显像

目的研究125I标记抗人CD40单克隆抗体(mAb)5H6同卵巢癌细胞HO8910体外结合的生物学特性以及在荷瘤鼠体内的分布和放射免疫显像.方法氯胺-T法进行mAb 5H6125I标记(125I-5H6),Lindmo法计算125I-5H6的免疫活性分数;细胞结合饱和实验进行Scatchard分析计算解离常数Kd及最大结合位点数Bmax;建立荷人卵巢癌(HO8910)裸鼠模型,分析125I-5H6在体内的分布,并用SPECT进行放射免疫显像.结果mAb 5H6标记率为(85.4±5.2)%,放射化学纯度为(99.2±0.5)%,125I-5H6免疫活性分数为(38.6±5.4)%,与HO8910细胞的亲和力Kd=(0.711±0.06)nmol/L.在荷瘤鼠体内特异性结合HO8910肿瘤,48小时达到高峰,放射免疫显像肿瘤清晰可见.结论125I-5H6同卵巢癌HO8910细胞在体外结合具有很高亲和力,在体内能特异性结合HO8910肿瘤,能获得优质的放射免疫图像.     

Determination of gene organization in the human IGHV region on single chromosomes

Organization of the IGHV genes (n=108) on single human chromosomes has been determined by detecting these sequences in single sperm using multiplex PCR amplification followed by microarray detection. A total of 374 single sperm samples from five Caucasian males were studied. Three deletion/insertion polymorphisms (Del I-Del III) with deletion allele frequencies ranging from 0.1 to 0.3 were identified. Del I is a previously reported polymorphism affecting three IGHV genes (IGHV1-8, IGHV3-9, and IGHV2-10). Del II affects a region 2-18 kb containing two pseudogenes IGHV(II)-28.1 and IGHV3-29, and Del III spans approximately 21-53 kb involving genes IGHV4-39, IGHV7-40, IGHV(II)-40-1, and IGHV3-41. Deletion alleles of both Dels II and III were found in a heterozygous state, and therefore, could not be easily detected if haploid samples were not used in the study. Results of the present study indicate that deletions/insertions together with other possible chromosomal rearrangements may play an important role in forming the genetic structure of the IGHV region, and may significantly contribute to antibody diversity. Since these three polymorphisms are located within or next to the 3' half of the IGHV region, they may have an important role in the expressed IGHV gene repertoire during immune response.     

Emergence of influenza A H1N2 reassortant viruses in the human population during 2001

A recombinant vaccinia virus encoding rotavirus protein NSP3 driven by an internal ribosome entry site (IRES) from the encephalomyocarditis (EMC) virus was able to abate protein synthesis in BSC1 cells by 25-fold, with as much as 30% of the remaining protein synthesis being NSP3. Hence NSP3 shuts off host cell protein synthesis down to the level seen during rotavirus infection but is unable to prevent translation from EMC IRES-driven genes. This effect was abolished by deletions in the eIF4G-binding (aa 274-313) and the dimerization (aa 150-206) but not the viral mRNA-binding (aa 83-149) domains, supporting that NSP3 functions in vivo as a dimer. Binding of eIF4G by NSP3 has been implicated in interfering with mRNA 5'-3' circularization, hence such circularization is essential for translation in mammalian cells.     

High-performance liquid chromatographic analysis of the D4 receptor antagonist SCH 66712 in rat plasma

SCH 66712 is a potent and selective dopamine D4 receptor antagonist. An HPLC method was developed for the analysis of SCH 66712 in the plasma of rats, a species used for safety evaluation of this compound. The method involved solid-phase extraction on an ethyl cartridge and HPLC separation on a reversed-phase C8 column with quantitation using a fluorescence detector. The calibration curve was linear over a concentration range of 5-100 ng/ml. The limit of quantitation was 5 ng/ml, where the coefficient of variation (C.V.) was 2.9% and the bias was 6%. The precision of the method was satisfactory as indicated by an intra-day C.V. of < or = 4% and an inter-day C.V. of < or = 6%. The accuracy was also satisfactory as shown by an intra-day bias of < or = 8% and an inter-day bias of < or = 9%. The assay was shown to be sensitive, specific, accurate, precise, and reliable for use in pharmacokinetic or toxicokinetic studies.     

High-resolution analyses of two different classes of tumor cells in situ tagged with alternative histochemical marker genes.

To evaluate interactions of two different tumor cell classes during the establishment of micrometastases at the single-cell level, two different BALB/c 3T3 tumor cell derivatives were established that harbor different histochemical marker genes: bacterial lacZ in a EJ-Harvey ras transformant (abbreviated LZEJ cells) and human placental alkaline phosphatase (ALP) gene in a human c-sis transformant (APSI cells). Several different histochemical staining methods were evaluated, using the distinctiveness of lacZ and ALP gene activities, for identification of these cell classes singly or together in the lung after their intravenous injection into nude mice. LZEJ and APSI cells could readily be distinguished from each other after co-injection by using specific and sequential staining protocols of whole organs or sections; staining of host organ cells was minimized. Co-injection of the two tumor cell classes resulted in similar numbers of homogeneous microfoci in lungs of LZEJ or APSI cells within minutes after injection that persisted for several hours before clearance of most of them. Furthermore, a significant percentage of foci could be identified containing both classes of tumor cells on whole-organ or section evaluations; these cohabiting foci resisted clearance from lungs. Therefore, use of two different histochemical marker genes to tag different classes of tumor cells provides a powerful approach for determining their in situ co-localization, cooperation, or interference with the establishment and development of micrometastases, as well as an opportunity to evaluate gene regulation in situ at the single-cell level.     

齿龈内阿米巴的致病作用与致病机制的研究

在注射免疫抑制剂 1周后的大白鼠龈缘涂抹齿龈内阿米巴 (Emtamoebagingivalis ,E .g .) ,5天后 ,牙龈组织出现溃疡、牙周脓肿形成、脓液查见活E .g .、牙槽骨吸收等牙周炎病症。电镜术与生化分析发现 :E .g .伪足活跃、有丰富的溶酶体 ,所含水解酶与ACP显著较健康组高 (P <0 0 1) ,可使牙周组织溶解与受损。SOD较健康组显著性低 (P <0 0 1) ,MDA显著性增高 (P <0 0 1) ,说明E .g .感染产生较多氧自由基可使细胞膜受损 ,加上口腔共生菌的协同作用使免疫力低下的宿主发生牙周炎。     

内生致冷原对家兔内毒素性发热第二热相的影响

为验证内生致冷原(EC)能否影响内毒素(ET)性发热第二热相或热限水平,并确实脑脊液中cAMP水平是否与EC的降温作用有关,作者用90只新西兰兔进行实验。观察:①输注人尿或等量生理盐液对正常家免体温的影响,检测EC效应期血浆和脑脊液中cAMP的含量;②在第二热峰出现前输注人尿或生理盐液对第二热相的影响,检测EC效应期血浆及脑脊液中cAMP的含量。结果表明:①人尿明显降低正常家兔的直肠温度,而等量生理盐液则无此作用,且两者均引起血浆及脑脊液中cAMP浓度的明显下降,提示EC的降温作用与脑cAMP浓度下降可能无重要关系;②人尿(EC)抑制ET性发热第二热相的形成,从而降低热限水平,变双相热为单相热,同量生理盐水无此作用,两者都能降低血浆和脑脊液中cAMP的水平,但EC不及NS明显,表明EC抑制第二热相或降低热限水平的作用也与cAMP浓度变化无重要关系,作者推论cAMP不是ET性发热第二热相的唯一成因。     

Chromosome 11p15.5 regional imprinting: comparative analysis of KIP2 and H19 in human tissues and Wilms' tumors

The imprinted H19 gene is frequently inactivated in Wilms' tumors (WTs) either by chromosome 11p15.5 loss of heterozygosity (LOH) or by hypermethylation of the maternal allele and it is possible that there might be coordinate disruption of imprinting of multiple 11p15.5 genes in these tumors. To test this we have characterized total and allele- specific mRNA expression levels and DNA methylation of the 11p15.5 KIP2 gene in normal human tissues, WTs and embryonal rhabdomyosarcoma (RMS). Both KIP2 alleles are expressed but there is a bias with the maternal allele contributing 70-90% of mRNA. Tumors with LOH show moderate to marked reductions in KIP2 mRNA relative to control tissues and residual mRNA expression is from the imprinted paternal allele. Among WTs without LOH most cases with H19 inactivation also have reduced KIP2 expression and most cases with persistent H19 expression have high levels of KIP2 mRNA. In contrast to the extensive hypermethylation of the imprinted H19 allele, both KIP2 alleles are hypomethylated and WTs with biallelic H19 hypermethylation lack comparable hypermethylation of KIP2 DNA. 5-aza-2'-deoxycytidine (aza-C) increases H19 expression in RD RMS cells but does not activate KIP2 expression. These data indicate coordinately reduced expression of two linked paternally imprinted genes in most WTs and also suggest mechanistic differences in the maintenance of imprinting at these two loci.      

Increased endostatin/collagen XVIII expression correlates with elevated VEGF level and poor prognosis in hepatocellular carcinoma.

Liver is the primary source for collagen XVIII, the precursor of angiogenesis inhibitor, endostatin. However, the role of endostatin/collagen XVIII expression during liver carcinogenesis remains elusive. Therefore, we studied its expression in five hepatoma cell lines and 105 hepatocellular carcinoma specimens. The poorly differentiated hepatoma cell lines exhibited increased endostatin/collagen XVIII levels compared with the well-differentiated ones. In hepatoma tissues, endostatin/collagen XVIII expression was detected in various types of liver cells and was significantly stronger in adjacent nontumor tissues than that in tumors (P<0.001). Endostatin/collagen XVIII expression in nontumor tissues correlated with tumor stages (P=0.014) and expression of vascular endothelial growth factor (P=0.007), but not the stages of hepatic fibrosis (P>0.05). Kaplan-Meier analysis showed that patients with higher endostatin/collagen XVIII expression had significantly shorter overall survival (P=0.011) and disease-free survival (P=0.0034). Moreover, endostatin/collagen XVIII level was an independent prognostic factor for tumor recurrence (P=0.034) by multivariate analysis. In conclusion, increased endostatin/collagen XVIII expression correlated with hepatoma progression and predicted poor prognosis for patients with hepatocellular carcinoma.