Gene therapy of malignant brain tumors: a rat glioma line bearing the herpes simplex virus type 1-thymidine kinase gene and wild type retrovirus kills other tumor cells.

Tumor cells infected with a retrovirus vector (VIK) containing the herpes simplex virus thymidine kinase (HSV-TK) gene can be selectively killed by treatment with nucleoside analogues, such as ganciclovir. To mediate delivery of the HSV-TK gene to "recipient" tumor cells, "donor" C6 rat glioma cells infected with the VIK vector (C6VIK) were superinfected with wild type Moloney murine leukemia virus (WT Mo-MLV). These modified donor cells (C6VIKWT) produced both wild type retrovirus and the VIK vector. In culture, C6VIKWT cells were 300-fold more sensitive to the toxicity of ganciclovir than were C6VIK cells, suggesting that the presence of wild type retrovirus contributed to the toxicity. Co-culture of C6VIKWT cells with the C6 subline, C6BAG, sensitized the latter to ganciclovir treatment. Nude mice inoculated subcutaneously with a mixture of C6VIKWT and C6BAG cells showed regression of subsequent tumors when treated with ganciclovir. The observations show that tumor cells modified in culture by infection with a retrovirus bearing the HSV-TK gene and wild type retrovirus are not only sensitive to ganciclovir, but can transfer this sensitivity to neighboring "naive" tumor cells in culture and in vivo.     

Mutation analysis in 16 patients with mtDNA depletion

Sixteen unrelated Southern European patients with the mitochondrial depletion syndrome (MDS) were analyzed for mutations in the TK2 and DGUOK genes. Three novel mutations were identified in TK2 (R183G, R254X, and 142insG). When we analyzed additional genes involved in the dNTPs pool, such as SLC25A19 (DNC) and NT5M (d-NT2), we did not detect mutations. The current study suggest that scanning the TK2, DGUOK, SLC25A19, and NT5M genes is likely to help about 10% of MDS families in terms of genetic counseling. Also, our findings indicate that genotype-phenotype correlations are not straightforward in MDS.     

自杀基因质粒表达载体pcDNA3.1(-)CMV.TK在鼻咽癌细胞中的构建及转染研究

目的 构建含TK自杀基因的质粒表达载体并进行转染研究。方法 根据已发表的Hsv-TK基因的核苷酸序列，设计并合成一对引物，以含TK基因的PGEM/TK质粒为模板扩增出TK基因全长CDS序列，将其克隆到质粒表达载体pcDNA3．1(-)CMV中，进行序列分析和酶切鉴定后，运用电穿孔法将重组体pCDNA3．1(-)CMV-CD车专人鼻咽癌CNE-2细胞中，观察其表达情况以及无毒前体药物GCV干预下对CNE-2细胞生长的抑制作用。结果 酶切和序列分析证明pcDNA3．1(-)(2MV．TK含完整的TK基因序列，RT-PCR从转染细胞总RNA中扩出预期片段；鼻咽癌CNE-2细胞在无毒前体药物GCV干预下，其生长受到抑制。结论 成功构建了质粒载体pcDNA3．1(-)CMVTK，可以将其作为鼻咽癌自杀基因治疗的一种载体。     

TK6和WTK1细胞tk位点突变敏感性的比较研究

目的比较研究2种起源于人类的tk^ /-杂合子细胞TK6和WTK1细胞对化合物——甲基磺酸甲酯(MMS)诱发tk位点突变的敏感性，为tk位点突变敏感细胞株的筛选提供实验依据。方法用标准诱变剂MMS处理TK6和WTK1细胞，对培养物进一步作tk位点突变测试，以及细胞p53基因蛋白表达水平的检测。结果MMS可诱导TK6和WTK1细胞tk位点的突变，其诱发突变分别是自发突变的2—7倍和3～10倍。WTK1细胞对MMS的细胞毒作用具有较大的抗性。MMS的作用下，WTK1细胞的突变频率分别是TK6细胞的15．7、19．0和20．4倍。在tk位点诱发了2种不同表型的突变集落，但以慢生长突变体为主。无论是自发突变还是MMS的诱发突变，WTK1细胞的突变频率均显著地高于TK6细胞。经MMS处理后，TK6细胞p53蛋白的表达水平增高更为明显。结论WTK1细胞是更为敏感的tk基因突变试验检测细胞株。MMS诱发的突变有染色体畸变和基因突变，但以染色体畸变为主。     

Interpreting in vitro developmental toxicity test battery results: The consideration of toxicokinetics

In the EU collaborative project ChemScreen an alternative, in vitro assay-based test strategy was developed to screen compounds for reproductive toxicity. A toxicokinetic modeling approach was used to allow quantitative comparison between effective concentrations in the in vitro test battery and observations of developmental toxicity in vivo. This modeling strategy is based on (1) the definition of relevant observations of toxicity in vivo, (2) simulation of the corresponding systemic concentrations in vivo by toxicokinetic modeling, and (3) correction for differences in protein binding and lipid partitioning between plasma and in vitro test media. The test results of a feasibility study with a number of known reproductive toxicants has been described previously (Piersma et al. [15]). In the present paper, we take a more detailed look at the toxicokinetics of these compounds, and add the analysis of some compounds from subsequent studies. We discuss how the consideration of toxicokinetics allowed comparison between test systems with differing test medium composition, has helped to interpret the in vitro findings in light of in vivo observations, and to gain confidence in the predictive value of the test battery outcomes. The same toxicokinetic modeling strategy, in reverse order, can now be used for risk assessment purposes to predict toxic doses in vivo from effective concentrations in vitro.     

TK1联合多种血清肿瘤标志物检测对胃癌诊断意义

目的 评价血清肿瘤标志物细胞质胸苷激酶也称为胸苷激酶1（thymidine kinase 1,TK1）、癌胚抗原（CEA）、糖蛋白抗原19-9（CA19-9）、糖蛋白抗原72-4（CA72-4）及糖蛋白抗原242（CA242）联合检测对胃癌诊断价值.方法 采用免疫印迹发光法分别检测45例胃癌患者、40例良性胃病患者以及40例健康人血清中TK1、CEA 、CA 19-9 、CA72-4和CA242的水平,比较三组间的差异.结果 胃癌组患者血清中TK1、CEA 、CA 19-9 、CA72-4和CA242的阳性率均显著高于良性胃病组及对照组（P＜0.05）.5项标志物联合检测的敏感性为82.1％,与单项检测相比差异有显著性（P＜0.05）.结论 TK1、CEA、CA19-9、CA72-4和CA242对胃癌具有较高的辅助诊断价值,5项标志物联合检测有助于提高胃癌诊断的敏感性.     

Enhancing the sensitivity of the thymidine kinase assay by using DNA repair-deficient human TK6 cells

The OECD guidelines define the bioassays of identifying mutagenic chemicals, including the thymidine kinase (TK) assay, which specifically detects the mutations that inactivate the TK gene in the human TK6 lymphoid line. However, the sensitivity of this assay is limited because it detects mutations occurring only in the TK gene but not any other genes. Moreover, the limited sensitivity of the conventional TK assay is caused by the usage of DNA repair-proficient wild-type cells, which are capable of accurately repairing DNA damage induced by chemicals. Mutagenic chemicals produce a variety of DNA lesions, including base lesions, sugar damage, crosslinks, and strand breaks. Base damage causes point mutations and is repaired by the base excision repair (BER) and nucleotide excision repair (NER) pathways. To increase the sensitivity of TK assay, we simultaneously disrupted two genes encoding XRCC1, an important BER factor, and XPA, which is essential for NER, generating XRCC1 ^−/−/XPA ^−/− cells from TK6 cells. We measured the mutation frequency induced by four typical mutagenic agents, methyl methane sulfonate (MMS), cis-diamminedichloro-platinum(II) (cisplatin, CDDP), mitomycin-C (MMC), and cyclophosphamide (CP) by the conventional TK assay using wild-type TK6 cells and also by the TK assay using XRCC1 ^−/−/XPA ^−/− cells. The usage of XRCC1 ^−/−/XPA ^−/− cells increased the sensitivity of detecting the mutagenicity by 8.6 times for MMC, 8.5 times for CDDP, and 2.6 times for MMS in comparison with the conventional TK assay. In conclusion, the usage of XRCC1 ^−/−/XPA ^−/− cells will significantly improve TK assay.     

Gene therapy targeted to telomerase in HCC by AF-hTERT-TK/GCV

Objective： The aim of this study was to observe the affection of targeted therapy to plasmid AF-pGL3-hTERT-TK in HCC cell line HepG2. Methods： We constructed therapeutic plasmid pGL3-hTERT-TK （containing suicide gene TK that promoted by promoter of hTERT） and was conjugated with AF-liposome （a protein that can combine with the receptor ASPGR on HCC cell surface）. Then we transfected HCC cell line HepG2 and hepatic cell L02 with AF-pGL3-hTERT-TK, observed the effects of therapeutic plasmid AF-pGL3-hTERT-TK for HCC cell line growth and apoptosis in vitro by Flow cytometry and Tun- nel method. Results： Our results showed that TK gene was 1100 bp in plasmid pGL3-hTERT-TK. Plasmid pGL3-hTERT-TK can effectively transfect HCC cell HepG2 and the transfection rate was 8.91%. By recognizing and combining effects of recep- tor protein ASPGR on HCC cell surface the therapeutic plasmid AF-pGL3-hTERT-TK could easily enter into HCC cell HepG2 and induce its apeptosis. The apoptosis rate was 85.87% while only 8.65% in L02 cell. Four days after AF-pGL3-hTERT-TK transfected HepG2 was intervention by ganciclovir （GCV）, a lot of apeptotic bodies were found by Tunnel analysis, while little apoptotic body was found in hepatic cell L02. Conclusion： AF-pGL3-hTERT-TK can target to HCC cell line and induce it to apoptesis, almost has no influence on hepatic cell L02. AF-pGL3-hTERT-TK has the potential therapeutic effects for HCC.     

Ethnoveterinary knowledge in pastoral Karamoja,Uganda

Ethnopharmacological relevance

The people of Karamoja of northern Uganda chiefly rely on ethnoveterinary knowledge (EVK) to control common livestock health problems. In spite of cattle's central role in Karamojong culture and livelihoods, there has been no systematic recording of their ethnoveterinary plant-based cures to date.

Aim of study

To document the remedies used to treat livestock diseases, their preparation and administration.

Methods

Data were collected using semi-structured interviews, guided questionnaires, group discussions, direct observations and collection trips.

Results

We present information on 209 plant species and 18 non-plant materials. Plant species are distributed over 116 genera and 54 families. The most common medicinal use was treatment against anaplasmosis. Balanites aegyptiacus, Carissa spinarum, Warburgia salutaris and Harrisonia abyssinica had the most uses of all species. All different plant parts were used; bark and underground parts were exploited more frequently than other plant parts. Most remedies listed used a single ingredient, typically soaked in water; only 12.8% remedies used multiple plants. The route of administration was primarily oral followed by topical applications. Almost all plants are collected from the wild; none of the few cultivated plants used had been planted for medicinal purposes.

Conclusions

The pastoralists in the study site possess a wealth of EVK which they use to maintain animal health. Their rich knowledge and high diversity of plants were recorded here for the first time.