Galactosylated DNA lipid nanocapsules for efficient hepatocyte targeting

The main objective of gene therapy via a systemic pathway is the development of a stable and non-toxic gene vector that can encapsulate and deliver foreign genetic materials into specific cell types with the transfection efficiency of viral vectors. With this objective, DNA complexed with cationic lipids of DOTAP/DOPE was encapsulated into lipid nanocapsules (LNCs) forming nanocarriers (DNA LNCs) with a size suitable for systemic injection (109 ± 6 nm). With the goal of increasing systemic delivery, LNCs were stabilised with long chains of poly(ethylene glycol) (PEG), either from a PEG lipid derivative (DSPE-mPEG₂₀₀₀) or from an amphiphilic block copolymer (F108). In order to overcome internalisation difficulties encountered with PEG shield, a specific ligand (galactose) was covalently added at the distal end of the PEG chains, in order to provide active targeting of the asialoglycoprotein-receptor present on hepatocytes. This study showed that DNA LNCs were as efficient as positively charged DOTAP/DOPE lipoplexes for transfection. In primary hepatocytes, when non-galactosylated, the two polymers significantly decreased the transfection, probably by creating a barrier around the DNA LNCs. Interestingly, galactosylated F108 coated DNA LNCs led to a 18-fold increase in luciferase expression compared to non-galactosylated ones.     

The GAL genetic switch: visualisation of the interacting proteins by split-EGFP bimolecular fluorescence complementation

A split-EGFP bimolecular fluorescence complementation assay was used to visualise and locate three interacting pairs of proteins from the GAL genetic switch of the budding yeast, Saccharomyces cerevisiae. Both the Gal4p-Gal80p and Gal80p-Gal3p pairs were found to be located in the nucleus under inducing conditions. However, the Gal80p-Gal1p complex was located throughout the cell. These results support recent work establishing an initial interaction between Gal3p and Gal80p occurring in the nucleus. Labelling of all three protein pairs impaired the growth of the yeast strains and resulted in reduced galactokinase activity in cell extracts. The most likely cause of this impairment is decreased dissociation rates of the complexes, caused by the essentially irreversible reassembly of the EGFP fragments. This suggests that a fully functional GAL genetic switch requires dynamic interactions between the protein components. These results also highlight the need for caution in the interpretation of in vivo split-EGFP experiments.     

Tethered spheroids as an in vitro hepatocyte model for drug safety screening

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.     

补肾调冲方对半乳糖致卵巢早衰大鼠性激素水平和卵巢组织形态学的影响

目的观察补肾调冲方对半乳糖致卵巢早衰(POF)大鼠性激素水平和卵巢组织形态学的影响。方法将20只成年雌性SD大鼠与15只同品系雄性大鼠合笼,运用半乳糖毒性法制造POF大鼠动物模型。雌性幼鼠产后第70 d(PND 70),选取标准饲料组动情周期为4～5 d的大鼠20只,设为空白对照组;选取半乳糖饲料组动情周期>7 d的大鼠40只,并将40只POF大鼠随机分为模型对照组与补肾调冲方治疗组,各20只。于PND 80开始,补肾调冲方治疗组大鼠每日灌服补肾调冲方水溶液6.26 g/kg,模型对照组及空白对照组大鼠每日分别灌服等容积0.9%氯化钠注射液,各组大鼠均连续灌胃4周。记录各组大鼠体质量增长情况及动情周期,检测血清性激素水平,同时进行卵巢组织形态学变化比较,光镜下计数各级卵泡数目。结果补肾调冲方可缩短补肾调冲方治疗组POF大鼠的动情周期;使增高的卵泡刺激素(FSH)、黄体生成激素(LH)水平下降,促进雌二醇(E2)分泌增加;对补肾调冲方治疗组POF大鼠卵巢组织有恢复作用;增加始基卵泡、窦前卵泡及窦状卵泡和黄体,减少闭锁卵泡。结论补肾调冲方对性激素的分泌有调节作用,可恢复卵巢功能,促进卵泡的发育和排卵。     

Identification of new galactose oxidase genes in Fusarium spp.

Galactose oxidase (GO) converts galactose to an aldehyde and has several biotechnological applications, including cancer diagnosis. It is mainly produced by Fusarium austroamericanum but is also produced by Fusarium acuminatum and by isolates of the Fusarium graminearum and Gibberella fujikuroi complexes. The F. austroamericanum GO gaoA gene has been cloned, but the GO genes from other secreting species have not been characterized. Problems associated with the F. austroamericanum GO such as high pI and low catalytic efficiency and thermostability, and the difficult purification process makes the search for homologous genes attractive. In this work, the GO genes from Fusarium verticillioides and Fusarium subglutinans, two species of the G. fujikuroi complex, were cloned, sequenced, and analyzed. New GO genes were found in databases and were used to construct a phylogenetic tree, which revealed the existence of three orthologous lineages of GO genes in Fusarium spp. In addition, RT‐PCR analyses revealed that the new GO cloned gene may be endogenously expressed in F. subglutinans but not in F. verticillioides, in the used culture conditions. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

三七皂苷单体Rg1对D-半乳糖模型鼠学习记忆和免疫功能的影响

目的 :探讨三七皂苷单体 Rg1对 D-半乳糖 ( D- gal)所致衰老模型鼠学习记忆行为和免疫功能的影响。方法 :Balb/c小鼠随机分为 4组 :衰老模型组、正常对照组、实验 组和实验 组。应用跳台和 Y-型迷路方法测定 D-半乳糖衰老模型鼠学习记忆行为 ;采用 3 H- Td R掺入法测定小鼠胸腺、脾细胞的增殖能力 ;生物活性检测法测定 IL- 2和 TNF- α水平的变化。结果 :三七皂苷单体 Rg1能明显提高衰老模型鼠对电刺激的逃避能力 ,改善 D- gal所致学习记忆能力下降。同时三七皂苷Rg1能明显增强衰老模型鼠胸腺和脾细胞增殖能力 ,对 IL- 2水平有所提高 ,血清 TNF- α的细胞毒作用较模型组降低。结论 :三七皂苷单体 Rg1能改善衰老模型鼠学习记忆能力并调节免疫功能。     

D-半乳糖对幼鼠心脏成纤维细胞拟衰老作用的实验研究

目的：研究D-半乳糖（D-gal）对心脏成纤维细胞拟衰老的诱导作用，探讨D-gal诱导心脏成纤维细胞衰老的机制。方法：不同浓度的D-gal作用于幼鼠心脏成纤维细胞，在不同的作用时间，采用MTT、细胞周期、超氧化物歧化酶（SOD）活力、丙二醛（MDA）含量等指标研究D-半乳糖对心脏成纤维细胞的作用。结果：D-gal组细胞活力明显低于对照组；增殖速度慢于对照组；细胞中DNA含量低于对照组，细胞周期S、G₂-M期减少，G₀-G₁期增加；心脏成纤维细胞中SOD活力低于对照组，MDA含量高于对照组。结论：提示D-gal对幼鼠心脏成纤维细胞的老化具有诱导作用。     

Improvement of quantitative testing of liver function in patients with chronic hepatitis C after installment of antiviral therapy

AIM: To investigate if and to what extent antiviral therapy influenced a broad panel of quantitative testing of liver function (QTLF). METHODS: Fifty patients with chronic hepatitis C were either treated with interferon (n=8), interferon/ribavirin (n=19) or peg-interferon/ribavirin (n=23). Quantitative testing of liver function, including aminopyrine breath test (ABT), galactose elimination capacity (GEC), sorbitol clearance (SCI) and indocyanine green clearance (ICG) was performed before and 3 mo after initiation of antiviral therapy. RESULTS: After 3 mo of antiviral treatment, 36 patients showed normal transaminases and were negative for HCV-RNA, 14 patients did not respond to therapy. ABT and GEC as parameters of microsomal and cytosolic liver function were reduced in all patients before therapy initiation and returned to normal values in the 36 therapy responders after 3 mo. Parameters of liver perfusion (SCI and ICG) were not affected by antiviral therapy. In the 14 non-responders, no changes in QTLF values were observed during the treatment period. CONCLUSION: ICG and SCI remained unaffected in patients with chronic hepatitis C, while ABT and GEC were significantly compromised. ABT and GEC normalized in responders to antiviral therapy. Early determination of ABT and GEC may differentiate responders from non-responders to antiviral treatment in hepatitis C.     

Partial remission of resistant nephrotic syndrome after oral galactose therapy

Focal segmental glomerulosclerosis is sometimes associated with a circulating permeability factor. It was proposed that this factor interacts with the sugars of the glycocalyx, and its high affinity for galactose was shown on the basis of chromatographic studies. Galactose inactivates it and seems to lead to its clearance from plasma. A toddler with a nephrotic syndrome resistant to corticosteroids was admitted. A renal biopsy revealed minimal change disease with deposition of immunoglobulin M. Immunosuppressive therapy with pulses of cyclophosphamide, low-dose combination immunosuppressive therapy, and later with mycophenolate mofetil failed to induce remission. A renal biopsy six years later showed transformation to FSGS. After unsuccessful treatment with monthly pulses of cyclophosphamide, we began therapy with tacrolimus, which showed no effect. After two months, we added oral galactose to tacrolimus for one month, after which proteinuria decreased by 50%. Seven months later, galactose was again added for six months, after which proteinuria remained below 2 g/24 h and the plasma albumin and cholesterol concentrations normalized. An adolescent girl with a nephrotic syndrome resistant to corticosteroids was admitted. A renal biopsy revealed mesangioproliferative glomerulonephritis with C1q nephropathy. Therapy with tacrolimus failed to induce remission. After six months, we added galactose for three months, which reduced proteinuria to 0.76 g/24 h. After the discontinuation of galactose therapy, proteinuria increased to 2.48 g/24 h, despite further treatment with tacrolimus. It seems that oral galactose at a dose of 0.2 g/kg twice a day could be a promising new and nontoxic therapy for the treatment of resistant nephrotic syndrome.     

半乳糖脂修饰的脂质体靶肝特性实验研究

目的研究用半乳糖脂修饰的脂质体与肝特异性半乳糖受体结合的能力，为用该脂质体包埋药物进行肝病治疗和肝造影提供依据。方法半乳糖经乙酰化、溴化、缩合、亲核取代反应制备半乳糖新糖脂质并与其他脂质制备脂质体，在二氯化锡还原作用下用９９ｍＴｃ标记脂质体，进行动物体内靶肝实验，用γ计数器检测各脏器放射性，计算摄取率。结果标记脂质体的标记率大于９５％，动物体内实验肝最大摄取率为８０１％，对照组为３１５％。乳糖封闭受体后，肝摄取率明显下降（３２１％），差异有显著意义（ｔ＝５２２，Ｐ＜００１）。结论半乳糖脂修饰的脂质体具有很好的靶肝作用，这种靶向性是与肝半乳糖受体结合的结果。