Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.     

Quantitative determination of the antitumor alkyl ether phospholipid edelfosine by reversed-phase liquid chromatography-electrospray mass spectrometry: application to cell uptake studies and characterization of drug delivery systems

Edelfosine is a synthetic alkyl ether phospholipid that represents a promising class of antitumor agents. However, analytical methods to measure these type compounds are scarce. The lack of a reliable methodology to quantify edelfosine is a major problem in ongoing and scheduled preclinical and clinical trials with this drug. We evaluated the applicability of high-performance liquid chromatography-mass spectrometry to determine edelfosine in biological samples and polymeric delivery systems. Sample pre-treatment involved polymer precipitation or cell lysis with methanol. HPLC separation was performed on an Alltima RPC(18) narrow-bore column and edelfosine quantification was done by electrospray ionization mass spectrometry (ESI-MS) using positive ion mode and selected ion monitoring. Assays were linear in the tested range of 0.3-10 microg/ml. The limit of quantification was 0.3 ng/sample in both matrices, namely biological samples and polymeric delivery systems. The interassay precision ranging from 0.79 to 1.49%, with relative errors of -6.7 and 12.8%. Mean extraction recovery was 95.6%. HPLC-ESI-MS is a reliable system for edelfosine analysis and quantification in samples from different sources, combining advantages of full automation (rapidity, ease of use, no need of extensive extraction procedures) with high analytical performance and throughput.     

EV71感染抗病毒药物及疫苗研究进展

手足口病在世界多个地区,尤其是亚洲爆发并流行,且其感染率和死亡率逐年增高,危害十分严重。肠道病毒71(Enterovirus 71,EV71)是手足口病(Hand,foot,andmouth disease,HFMD)的主要病原体,以感染婴幼儿为主,其感染常伴随神经系统并发症,严重可导致儿童死亡。近年来,分子生物学和抗病毒研究方面取得的进展为EV71感染的预防及治疗提供了新的途径。本文对EV71病毒学特点及抗EV71药物的筛选、疫苗开发、RNA干扰等进行了综述,以期为相关研究提供参考。     

禽流感治疗药物研究进展

禽流感是由正黏液病毒科甲型流感病毒引起的对人类健康和社会发展构成极大威胁的烈性传染病,高致病性禽流感暴发突然,具有极高的发病率和死亡率。目前具有确切疗效的抗禽流感治疗药物品种很少,公认的药物只有奥塞米韦,此外流感病毒的抗药性也是一个重要的问题,近年来出现的甲型H1N1病毒更给人类敲响了警钟,因此研究更多的治疗药物和治疗手段对于禽流感的防控十分必要。从禽流感治疗化学药物和生物药物几个方面对禽流感治疗研究进展进行了综述。     

Efficacy of antifungal agents in tissue conditioners in treating candidiasis

Chronic atrophic candidiasis is prevalent in up to 72% of institutionalized geriatric populations and is causally associated with Candida albicans. Topical antifungal treatments are difficult to implement in some geriatric patients due to cognitive impairment, reduced motor dexterity and memory loss. Objective: This in vitro study incorporated antifungal agents into tissue conditioners to investigate the effectiveness of this method of drug delivery. Design: Combinations of nystatin, fluconazole, itraconazole and Coe Soft, Viscogel, Fitt were tested at 1, 3, 5, 7, 9 and 11wt/wt%, with and without sterilized saliva. 6 mm diameter cores were punched in Sabouraud plates pre-grown with standardized C. albicans. Antifungal agents plus tissue conditioner mixtures were injected into each core. Inhibition diameters were measured for 14 days. Results: Cores with only tissue conditioners acted as negative control and showed no significant inhibition activity (ANOVA, p>0.05). Peak activity was between 65 to 89 hours; followed by a plateau. Itraconazole had greater fungicidal activity than fluconazole; while nystatin was found to have the least fungicidal activity (ANOVA. p<0.05). The most effective concentration for nearly all combinations was 5%wt/wt (ANOVA, p<0.05). Specimens with saliva showed greater antifungal activity than those without (t-test. p<0.001). Itraconazole altered the physical properties of Viscogel hence this combination is not recommended for clinical use. Conclusion: The treatment of chronic atrophic candidiasis by incorporation of antifungal drugs into tissue conditioners is efficacious. 5% wt/wt itraconazole mixed with Coe Soft or Fitt is recommended for clinical study where compliance of patient or care giver cannot be relied upon. Peak antifungal activity at 3 days suggests that mixtures prepared for clinical study may be replaced soon after this time for maximum effectiveness.     

Increased expression of rat synuclein in the substantia nigra pars compacta identified by mRNA differential display in a model of developmental target injury

Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.     

Microbiological Characteristics and Susceptibility Patterns of Strains of Rhodotorula Isolated from Clinical Samples

The members of the genus Rhodotorula show a marked ubiquity. In man, they have been isolated from faeces, nails, skin, sputum, digestive tract and adenoids, forming part of the normal human flora, although in recent years cases have been reported of both local and systemic infection by this yeast. There are virtually no studies in the literature on the sensitivity of this genus to the antifungal agents in common clinical use. Therefore, it is considered of interest to study the microbiological characteristics and the susceptibility patterns of Rhodotorula isolated from clinical samples. A total of 35 different strains of Rhodotorula were studied. In vitro susceptibility testing to 5-fluorocytosine, amphotericin B, ketoconazole, fluconazole and itraconazole was performed. All the strains were considered sensitive to 5-fluorocytosine, amphotericin B, ketoconazole and itraconazole and resistant to fluconazole. As a conclusion, we can state that all the antifungal agents tested, except fluconazole, are useful medicaments for the treatment of infections by the Rhodotorula genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.     

Effect of Anti-Microtubular Agents on Respiration and Ultrastructural Organization of Wheat Leaf Cells

The effect of anti-microtubular agents oryzalin (15 M) and colchicine (1 mM) on respiration and fine cellular organization of leaves was studied in wheat (Triticum aestivum L.) seedlings. Unlike oryzalin, colchicine considerably suppressed total respiration and the activities of cytochrome and cyanide-resistant pathways of electron transport, probably due to its rotenone-like effect since the latter was negated by vicasol that shunts the first complex of respiratory chain. Oryzalin-induced changes in the ultrastructure of mitochondria and polysomes corresponding to a decrease in their functional activity were detected. The changes in the shape of mitochondria were observed in the companion cells, whereas the mesophyll organelles did not respond to oryzalin. Disintegration of polysomes induced by oryzalin was detected in the cells of both tissues. Association of microtubules with mitochondria, polysomes, and plasma membrane was detected in situ. These data suggest that the intact cytoskeleton may participate in the spatial organization of energy transformation and protein synthesis machinery of the cells; the fact that the observed changes were tissue-specific points at the functional role of microtubules and/or to the number of targets for the inhibitor.