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31.
Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes 总被引:3,自引:1,他引:3
Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347- bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596- bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35- , 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins. 相似文献
32.
Liu SC; Palek J; Yi SJ; Nichols PE; Derick LH; Chiou SS; Amato D; Corbett JD; Cho MR; Golan DE 《Blood》1995,86(1):349-358
Southeast Asian ovalocytosis (SAO) is an asymptomatic trait characterized by rigid, poorly deformable red cells that resist invasion by several strains of malaria parasites. The underlying molecular genetic defect involves simple heterozygous state for a mutant band 3 protein, which contains a deletion of amino acids 400 through 408, linked with a Lys 56-to-Glu substitution (band 3-Memphis polymorphism). To elucidate the contribution of the mutant SAO band 3 protein to increased SAO red blood cell (RBC) rigidity, we examined the participation of the mutant SAO band 3 protein in increased band 3 attachment to the skeleton and band 3 oligomerization. We found first that SAO RBC skeletons retained more band 3 than normal cells and that this increased retention preferentially involved the mutant SAO band 3 protein. Second, SAO RBCs contained a higher percentage of band 3 oligomer-ankyrin complexes than normal cells, and these oligomers were preferentially enriched by the mutant SAO protein. At the ultrastructural level, the increased oligomer formation of SAO RBCs was reflected by stacking of band 3-containing intramembrane particles (IMP) into longitudinal strands. The IMP stacking was not reversed by treating SAO RBCs in alkaline pH (pH 11), which is known to weaken ankyrin-band 3 interactions, or by removing the cytoplasmic domain of band 3 from SAO membranes with trypsin. Finally, we found that band 3 protein in intact SAO RBCs exhibited a markedly decreased rotational mobility, presumably reflecting the increased oligomerization and the membrane skeletal association of the SAO band 3 protein. We propose that the mutant SAO band 3 has an increased propensity to form oligomers, which appear as longitudinal strands of IMP and exhibit increased association with membrane skeleton. This band 3 oligomerization underlies the increase in membrane rigidity by precluding membrane skeletal extension, which is necessary for membrane deformation. 相似文献
33.
Effects of interleukin-11 on the proliferation and cell cycle status of myeloid leukemic cells 总被引:1,自引:0,他引:1
Interleukin-11 (IL-11) is a pleiotropic cytokine with effects on many different targets. Within the hematopoietic system, the effects of IL- 11 are largely manifest only through combination with other cytokines, including IL-3 and Steel factor (SF). In the present study, we addressed the question of IL-11 responsiveness within the different types of human leukemic cells, as well as the mechanism of action of IL- 11 at the cellular level. Analysis of a panel of samples from different patients with acute myeloblastic leukemia (AML) and myeloid leukemic cell lines indicated that IL-11 alone was ineffective in supporting myeloid leukemic cell growth but frequently enhanced growth supported by IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or SF. In contrast, three acute pre-B lymphocytic leukemia (pre-B-ALL) and two acute T lymphocytic leukemia (T-ALL) lines failed to respond to IL- 11 alone or when combined with other cytokines. The growth enhancement of IL-11 among the AML patient samples was dose dependent and remarkably constant with half-efficient concentrations in the range of 0.3 to 0.4 ng/mL. The thymidine suicide studies with the patient samples revealed that 40% to 50% of the blast cells were in S-phase when exposed for 16 hours to IL-3 and this level was increased to 70% to 90% in response to either IL-11 or IL-6. Our data suggest that the latter two interleukins act synergistically with the direct mitogenic factor, IL-3, in triggering AML blast-cell proliferation. Detailed analysis with several patient samples further revealed that SF and IL- 11 both enhance IL-3-supported clonogenic growth of AML blasts and the combination of all three growth factors yields optimal growth. In contrast, IL-6 does not further enhance the effect of IL-11. These results indicate that SF and IL-11 enhance IL-3-dependent clonogenic growth through two distinct pathways, whereas IL-6 and IL-11 may trigger the same pathway. 相似文献
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35.
Labetalol, a combined alfa and beta-adrenergic receptor antagonist, is used as an antihypertensive drug. We report a case of an acute rise in blood pressure and lower limb pain due to the inadvertent intrathecal administration of labetalol, mistaking it for bupivacaine, during obstetric anesthesia. The situation was rescued by converting to general anesthesia. The cesarean delivery was uneventful, and mother as well as newborn child showed no ill-effect. This particular medication error was attributable to a failure on the part of the doctors administering the injection to read and cross-check medication labels and the practice of keeping multiple injections together. In the absence of an organized medication error reporting system and action on that basis, such events may recur in future.KEY WORDS: Bupivacaine, labetalol, medication error, spinal anesthesia 相似文献
36.
Meenakshi Singh Abhijit Hazra Yogesh P. Bharitkar Ritu Kalia Ashutosh Sahoo Sneha Saha V. Ravichandiran Shekhar Ghosh Nirup B. Mondal 《RSC advances》2018,8(34):18938
Curcumin has been transformed to several diversely substituted bis-pyrrolizidino/thiopyrrolizidino oxindolo/acenaphthyleno curcuminoids via a sequential azomethine ylide cycloaddition reaction using isatins/acenaphthoquinone and proline/thioproline as the reagents. The products were separated via extensive chromatography and characterized by 1D/2D NMR and HRMS analysis.Curcumin has been transformed to several diversely substituted bis-pyrrolizidino/thiopyrrolizidino oxindolo/acenaphthyleno curcuminoids via a sequential azomethine ylide cycloaddition reaction. 相似文献
37.
38.
Jitendra Nath Pal Prahas Biswas Avik Roy Sunit Hazra Somnath Mahato 《Indian Journal of Orthopaedics》2015,49(4):408-417
Background:Functional brace application for isolated humeral shaft fracture persistently yields good results. Nonunion though uncommon involves usually the proximal third shaft fractures. Instead of polyethylene bivalve functional brace four plaster sleeves wrapped and molded with little more proximal extension expected to prevent nonunion of proximal third fractures. Periodic compressibility of the cast is likely to yield a better result. This can be applied on the 1st day of the presentation as an outpatient basis. Comprehensive objective scoring system befitting for fracture humeral shaft is a need.Results:The results were assessed using 100 point scoring system where union allotted 30 points and 60 points allotted for angulations (10), elbow motion (10), shoulder abduction (10), shortening (5), rotation (5), absence of infection (10), absence of nerve palsy during treatment (10). Remaining 10 points were allotted for five items with two points each. They were the absence of skin sore, absence of vascular problem, absence of reflex sympathetic dystrophy (RSD), recovery of paralyzed nerve during injury and recovery of paralyzed nerve during treatment. Results were considered excellent with 90 and above, good with 80–89, fair with 70–79 and poor below 70 point. Results at 6 months were excellent in 43.94% (n = 29), good in 42.42% (n = 28), fair in 9.1% (n = 6), poor in 4.55% (n = 3). Union took place in 98.48% (n = 65) with an average of 10.3 weeks (range 6–16 weeks). 87.5% (n = 7) paralyzed radial nerve recovered. All wounds healed. Four patients had transient skin problem. One patient with mid shaft fracture had nonunion due to the muscle interposition.Conclusion:Modified functional cast brace is one of the options in treatment for humeral shaft fractures as it can be applied on the 1st day of the presentation in most of the situations. Simple objective scoring system was useful particularly in uneducated patients. 相似文献
39.
Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer 总被引:2,自引:0,他引:2
Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA. 相似文献
40.