Biomedical subjects
Publications and source records attributed to G Qu.
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Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.
The central nervous system (CNS) is a predominant site of involvement in several lysosomal storage diseases (LSDs); and for many patients, these diseases are diagnosed only after the onset of symptoms related to the progressive accumulation of macromolecules within lysosomes. The mucopolysaccharidosis type VII (MPS VII) mice are deficient for the lysosomal enzyme beta-glucuronidase and, by early adulthood, develop a significant degree of glycosaminoglycan storage within neuronal, glial, and leptomeningeal cells. Using this animal model, we investigated whether gene transfer mediated by a recombinant adeno-associated virus (rAAV) vector is capable of reversing the progression of storage lesions within the CNS. Adult MPS VII mice received intracerebral injections of 4 X 10(7) infectious units of a rAAV vector carrying the murine beta-glucuronidase (gus-s(a)) cDNA under the transcriptional direction of the cytomegalovirus immediate-early promoter and enhancer. By 1 month after vector administration, transgene-derived beta-glucuronidase was present surrounding the injection site. Enzyme levels were between 50 and 240% of that found in wild-type mice. This level of beta-glucuronidase activity was sufficient to reduce the degree of lysosomal storage. Moreover, the reduction in storage was maintained for at least 3 months post-rAAV administration. These data demonstrate that rAAV vectors can transduce the diseased CNS of MPS VII mice and mediate levels of transgene expression necessary for a therapeutic response. Thus, rAAV vectors are potential tools in the treatment of the mucopolysaccharidoses and other lysosomal storage diseases.
The infrared (IR) and Raman spectra are reported for solutions of lithium perchlorate in propylene carbonate (PC), N,N-dimethylformamide (DMF) and PC + DMF mixtures. The band splittings of symmetric ring deformation for PC and O=CN deformation for DMF suggest that there is a strong interaction between lithium cations and solvent molecules. The solvent molecules have been assigned to two types, the free and complexed molecules. By a comparison of the intensity for the corresponding bands, it has been concluded that Li+ cations are preferentially solvated by DMF molecules in the LiClO4/PC-DMF solutions. This has been explained by the difference in values of donor number.
This paper studied the effect of nitrogen fertilizer on water use efficiency(WUE) and relevant parameters of two years old potted apple trees(Starkrimson/Malus hupenensis) under different soil moisture condition. The results showed that under adequate soil moisture, WUE was decreased with increasing application of nitrogen fertilizer. Nitrogen fertilizer application resulted in an increase of stomatal conductane. Consequently, the transpiration rate was increased more than photosynthetic rate did. Under soil drought, the WUE of plants applied with nitrogen fertilizer was apparently higher than that of control. The WUE value was in the order of high N > medium N > low N. The improvement of WUE was due to the increase of mesophyll capacity, which led to the promotion of photosynthesis.
Rearrangements in heterochromatin in the vicinity of the centromeres of chromosomes 1 and 16 are frequent in many types of cancer, including ovarian epithelial carcinomas. Satellite 2 DNA is the main sequence in the unusually long heterochromatin region adjacent to the centromere of each of these chromosomes. Rearrangements in these regions and hypomethylation of satellite 2 DNA are a characteristic feature of patients with a rare recessive genetic disease, ICF (immunodeficiency, centromeric region instability, and facial anomalies). In all normal tissues of postnatal somatic origin, satellite 2 DNA is highly methylated. We examined satellite 2 DNA methylation in ovarian tumors of different malignant potential, namely, ovarian cystadenomas, low malignant potential (LMP) tumors, and epithelial carcinomas. Most of the carcinomas and LMP tumors exhibited hypomethylation in satellite 2 DNA of both chromosomes 1 and 16. A comparison of methylation of these sequences in the three types of ovarian neoplasms demonstrated that there was a statistically significant correlation between the extent of this satellite DNA hypomethylation and the degree of malignancy (P<0.01). Also, there was a statistically significant association (P<0.005) between genome-wide hypomethylation and undermethylation of satellite 2 DNA among these 17 tumors. In addition, we found abnormal hypomethylation of satellite alpha DNA in the centromere of chromosome 1 in many of these tumors. Our findings are consistent with the hypothesis that one of the ways that genome-wide hypomethylation facilitates tumor development is that it often includes satellite hypomethylation which might predispose cells to structural and numerical chromosomal aberrations. Several of the proteins that bind to pericentromeric heterochromatin are known to be sensitive to the methylation status of their target sequences and so could be among the sensors for detecting abnormal demethylation and mediating effects on chromosome structure and stability.
Adeno-associated virus (AAV) is a promising vector for central nervous system (CNS) gene transfer, but a number of issues must be addressed if AAV is to be used for widespread delivery throughout the CNS. Our aim was to test the effect of dose, route of delivery, and hydroxyurea treatment on brain expression of beta-galactosidase activity after cerebral inoculation with an rAAV-lacZ vector (rAAV-beta-gal). We also wished to test whether an immune response appeared against the vector and the transgene product. We found in BALB/c mice that beta-Gal expression increased during the first 2 months after inoculation, then decreased slightly by 4 months, and continued out to 6, 12, and 15 months in single animals. Cerebral injection produced localized beta-Gal expression that did not diffuse to other regions despite a fivefold increase in injection volume. Intraventricular injection resulted in negligible transduction. Antibodies to AAV capsid protein and beta-Gal appeared at low levels at 2 and 4 months, but correlated poorly with beta-Gal expression and did not prevent readministration of rAAV-beta-gal. Hydroxyurea treatment did not result in increased transduction in vivo. We conclude that our study confirms rAAV vectors as having considerable potential for CNS gene transfer; however, several important problems must be addressed if this vector system is to be used for long-term transduction of the entire brain. Sustained, regulatable expression will be needed if rAAV is to be used in the treatment of chronic CNS disease. The difficulty in delivering AAV to diverse regions of the brain is an important problem that must be overcome if these vectors are to be used for anything beyond localized transduction.
Recombinant adeno-associated virus (rAAV) vectors have shown significant promise as vehicles for in vivo gene transfer, particularly for transduction of organs composed primarily of non-dividing cells (i.e., muscle, CNS, and liver). However, the mechanistic basis for this desirable property remains unclear. To investigate the fate of rAAV genomes in mouse brain, we stereotactically injected an rAAV vector carrying the E. coli lacZ gene into the caudate of BALB/c mice and demonstrate efficient transduction of mouse brain cells that possess cellular morphology consistent with post-mitotic neurons. We observed a significant increase in beta-galactosidase expression from 5 to 56 days after injection that paralleled the disappearance of single-stranded DNA input genomes. Analysis of in vivo viral DNA forms over time out to 5 months after inoculation revealed that rAAV genomes associated with high molecular weight mouse chromosomal DNA by 14 days after injection and persisted for the length of this study. The pattern of Southern hybridization was consistent with random viral integration in predominantly head-to-tail concatameric arrays. Importantly, we also documented an additional DNA species that appears to be a monomeric episomal circular form based on nuclease sensitivity assays. These data are the first to document the existence of multiple vector DNA forms present within the adult murine brain following direct rAAV inoculation and therefore, provide insight into the molecular events that ultimately result in long-term rAAV mediated transgene expression.
Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.
Fluorescence in situ hybridization (FISH) with microdissection probes from human chromosomes 3 and 6 was applied to visualize arm and subregional band domains in human amniotic fluid cell nuclei. Confocal laser scanning microscopy and quantitative three-dimensional image analysis showed a pronounced variability of p- and q-arm domain arrangements and shapes. Apparent intermingling of neighbouring arm domains was limited to the domain surface. Three-dimensional distance measurements with pter and qter probes supported a high variability of chromosome territory folding.
Mouse BC3H1 myogenic cells and a bi-functional chemical cross linking reagent were utilized to investigate the polymerization of newly-synthesized vascular smooth muscle (alpha-actin) and non-muscle (beta- and gamma-actin) actin monomers into native F-actin filament structures during myogenesis. Two actin dimer species were identified by SDS-PAGE analysis of phenylenebismaleimide-cross linked fractions of BC3H1 myoblasts and myocytes. P-dimer was derived from the F-actin-enriched, detergent-insoluble cytoskeleton. Pulse-chase analysis revealed that D-dimer initially was associated with the cytoskeleton but then accumulated in the soluble fraction of lysed muscle cells that contained a non-filamentous or aggregated actin pool. Immunoblot analysis indicated that non-muscle and smooth muscle actins were capable of forming both types of dimer. However, induction of smooth muscle alpha-actin in developing myoblasts coincided with an increase in D-dimer level which may facilitate actin stress fiber assembly. Smooth muscle alpha-actin was rapidly utilized in differentiating myoblasts to assemble extraction-resistant F-actin filaments in the cytoskeleton whereas non-muscle beta- and gamma-actin filaments were more readily dissociated from the cytoskeleton by an extraction buffer containing ATP and EGTA. The data indicate that cytoarchitectural remodeling in developing BC3H1 myogenic cells is accompanied by selective actin isoform utilization that effectively segregates multiple isoactins into different sub-cellular domains and/or supramolecular entities.
To search the relationships between urinary albumin excretion and blood vessel lesion in non-insulin-dependent diabetics, we compared the differences of blood-lipid, blood pressure, coronary heart disease and retina disease between 58 patients. The group with microalbuminuria had significantly lower concentration of apolipoprotein A, and apolipoprotein A1/apolipoprotein B100 than that of the group with normal albuminuria, but there was no significant change in concentrations of trigly cerides, total cholesterol, high density lipoprotein, and apolipoprotein B100. Coronary heart disease and retina disease were significantly increased in group with microalbuminuria. There was negative correlation with apolipoprotein A1 and value of apolipoprotein A1/apolipoprotein B100, but positive correlation with systolic blood pressure by linear regression analysis for urinary albumin excretion.
RATIONALE AND OBJECTIVES: We compared subjective and objective measures of imaging performance using variations of Rose- and Burger-type low-contrast phantoms with trained (radiology residents) and untrained (graduate students) observers. METHODS: With one phantom variant, observers indicated the total number of objects seen when test objects were presented in a regular pattern (subjective). With the second phantom variant, observers stated whether a low-contrast disk was present in each locale, thereby permitting the true-positive fraction and false-positive fraction to be determined (objective). RESULTS: The untrained-observer group had a significantly lower imaging performance than the trained observer group in subjective tests. These differences were not found on objective tests. For the trained-observer group, similar contrast levels were required in subjective and objective tests to yield a 50% rate of detection. CONCLUSION: Trained observers are superior subjects compared with untrained observers for assessing imaging performance using subjective low-contrast phantoms. In experiments using phantoms that allowed objective testing, both groups of observers yield similar results.
BACKGROUND: We conducted a retrospective cohort study of lung cancer among silicotics in China's metallurgical industries. The study cohort consisted of 4372 male silicotics diagnosed and alive before 1 January 1980. The follow-up period was 1 January 1980 to 31 December 1989. RESULTS: Standardized mortality ratios (SMR) of all deaths, all cancers, chronic bronchopneumonia, pneumonia and pulmonary heart disease, and pulmonary tuberculosis showed statistically significant excesses. The SMR for lung cancer for the total cohort was 237 (P < 0.01). When the total study cohort was divided into various categories by occupation (including iron ore mining, ore-sintering, refractory brick manufacturing, iron and steel smelting, and steel casting), significant excess lung cancer risk was observed among silicotics in all categories (SMR > 200, P < 0.01) except for casting (SMR = 157, P > 0.05). Excess lung cancer was not related to years since diagnosis of silicosis or years of exposure to silica. There was more than twofold excess lung cancer risk among both smoking (SMR = 257, P < 0.01) and non-smoking silicotics (SMR = 209, P < 0.01). Silicosis increased the risk of lung cancer.
Two flavone glycoside compounds were isolated from Sonchus arvensis by means of chemical and spectroscopic analysis. They were identified as quercetin-3-O-alpha-L-rhamnoside and kaempferol-3, 7-alpha-L-dirhamnoside.
Suicide is the leading cause of non-natural deaths in China. Female suicide rates are higher than male rates, and the rural suicide rate is 2.8 times higher than the urban rate. In 16,568 cases of suicide, the male to female ratios observed were 0.92 and 0.81 in urban and rural areas, respectively. The male to female ratio of the suicide rates in China is close to that of other Asian countries (1.2-1.5), but much lower than that in 26 European countries. In about two-thirds of all suicides, family disputes and love disappointments are the precipitants. The higher rate of suicide among women probably reflects the fact that Chinese women are of low social and economic position, and have few social resources to protect them, especially in remote areas.
Four Chinese hemophiliacs with HIV infection have been followed up in Zhejiang Province since 1985. A plan for optimal care of HIV seropositive patients was proposed, including surveillance and care; clinical follow-up and preventive education of asymptomatic HIV seropositive patients; keeping the disease confidential so as not to stir up unnecessary social unease; propaganda and health education about acquired immunodeficiency syndrome (AIDS); treatment with traditional Chinese medicines; and regular surveillance of family members and medical personnel. This plan is regarded as feasible and effective for the prevention and control of AIDS in China.
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