Human VEGF165-myoblasts produce concomitant angiogenesis/myogenesis in the regenerative heart

Bioengineering the regenerative heart may provide a novel treatment for heart failure. On May 14, 2002, a 55-year-old man suffering from ischemic myocardial infarction received 25 injections carrying 465 million cGMP-produced pure myoblasts into his myocardium after coronary artery bypass grafting. As on August 28, 2002, his EKG was normal and showed no arrhythmia. His ejection fraction increased by 13%. He no longer experienced shortness of breath and angina as he did before the treatment. Three myogenesis mechanisms were elucidated with 17 human/porcine xenografts using cyclosporine as immunosuppressant. Some myoblasts developed to become cardiomyocytes. Others transferred their nuclei into host cardiomyocytes through natural cell fusion. As yet others formed skeletal myofibers with satellite cells. De novo production of contractile filaments augmented the heart contractility. Human myoblasts transduced with VEGF₁₆₅ gene produced six times more capillaries in porcine myocardium than in placebo. Xenograft rejection was not observed for up to 20 weeks despite cyclosporine discontinuation at 6 weeks. Pros and cons of autografts vs. allografts are compared to guide future development of heart cell therapy. (Mol Cell Biochem 263: 173–178, 2004)     

Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila

The Drosophila RNase III enzyme Dicer-2 processes double-stranded RNA (dsRNA) precursors into small interfering RNAs (siRNAs). It also interacts with the siRNA product and R2D2 protein to facilitate the assembly of an RNA-induced silencing complex (RISC) that mediates RNA interference. Here, we characterized six independent missense mutations in the dicer-2 gene. Four mutations (P8S, L188F, R269W, and P365L) in the DExH helicase domain reduced dsRNA processing activity. Two mutations were located within an RNase III domain. P1496L caused a loss of dsRNA processing activity comparable to a null dicer-2 mutation. A1453T strongly reduced both dsRNA processing and RISC activity, and decreased the levels of Dicer-2 and R2D2 proteins, suggesting that this mutation destabilizes Dicer-2. We also found that the carboxyl-terminal region of R2D2 is essential for Dicer-2 binding. These results provide further insight into the structure-function relationship of Dicer, which plays a critical role in the siRNA pathway.     

Direct and nondestructive verification of PNA immobilization using click chemistry

The fluorogenic 1,3-Huisgen dipolar cycloaddition reaction was used as part of a novel immobilization strategy of PNA capture probes on a microarray. By using this click chemistry, azidocoumarin-anchored PNA probes were immobilized on phenyl acetylene-modified glass slides with the simultaneous generation of the fluorescent triazolylcoumarin moiety. Since the emitting moieties are generated in the immobilization reaction itself, fluorescent signals can be used to directly monitor the integrity of immobilization in a nondestructive manner. By using this strategy, PNA microarrays were prepared and successfully employed to perform microarray-based diagnosis of selected mutations in the breast cancer susceptibility gene BRCA1.     

Structure of the nucleoid-associated protein Cnu reveals common binding sites for H-NS in Cnu and Hha

Cnu is a nucleoid protein that has a high degree of sequence homology with Hha/YmoA family proteins, which bind to chromatin and regulate the expression of Escherichia coli virulence genes in response to changes in temperature or ionic strength. Here, we determined its solution structure and dynamic properties and mapped H-NS binding sites. Cnu consists of three alpha helices that are comparable with those of Hha, but it has significant flexibility in the C-terminal region and lacks a short alpha helix present in Hha. Upon increasing ionic strength, the helical structure of Cnu is destabilized, especially at the ends of the helices. The dominant H-NS binding sites, located at helix 3 as in Hha, reveal a common structural platform for H-NS binding. Our results may provide structural and dynamic bases for the similarity and dissimilarity between Cnu and Hha functions.     

Enantioselective determination of metoprolol and major metabolites in human urine by capillary electrophoresis

The enantiomeric separation of metoprolol and its metabolites in human urine was undertaken using capillary electrophoresis (CE). Resolution of the enantiomers was achieved using carboxymethyl-β-cyclodextrin (CM-β-CD) as the chiral selector. A 100-mM acetate buffer (pH 4.0) containing 5% 2-propanol and 10 mM CM-β-CD resulted in the optimum separation of the metoprolol enantiomers and its acidic metabolite in human urine. Following a single metoprolol oral administration of 100 mg racemic metoprolol tartrate, stereoselective pharmacokinetic analysis showed that urinary acidic metabolite 3 of metoprolol accounted for 62.3% of the dose with an R/S ratio of 1.23 and urinary unchanged metoprolol 1 accounted for 6.3% of the dose with an R/S ratio of 0.72.     

电穿孔介导质粒DNA肿瘤内转移抑制恶性肿瘤生长与转移

利用携带绿色荧光蛋白（green fluorescent protein, GFP）编码基因的表达质粒,测试电穿孔方法介导目的基因活体组织内转移的效率并优化电击参数.在此基础上采用电穿孔技术直接将编码白介素12（IL-12）、白介素2（IL-2）、粒单细胞克隆刺激因子（GM-CSF）等免疫调节因子或反义血管内皮细胞生长因子121（VEGF₁₂₁）、可溶性血管内皮细胞膜受体（sFlk-1及ExTek）等血管生成抑制因子表达质粒转移至肿瘤局部.实验结果表明电穿孔介导GFP表达质粒肌肉内转移的效率较高,GFP可在肌细胞内持续高水平表达3周以上,而在肿瘤细胞内只能表达4～6 d,但高电压短脉冲电击组肿瘤内GFP阳性细胞数比低电压长脉冲组高2.68倍.多次电击介导IL-12表达质粒转移至肿瘤组织内,可有效地抑制小鼠膀胱癌BTT-gfp、人乳腺癌MCF-7及肝癌SMMC 7721-gfp的生长.MCF-7对血管生成抑制因子基因转移治疗较敏感,单独应用反义VEGF₁₂₁、sFlk-1或ExTek即显示明确的治疗效果.SMMC 7721-gfp单独应用sFlk-1有效.小鼠膀胱癌对单独应用反义VEGF₁₂₁、sFlk-1或ExTek治疗效果不理想,但联合应用sFlk-1和ExTek仍然可以有效地抑制肿瘤生长与转移,甚至使肿瘤缩小或消失.提示电穿孔技术是一项高效、安全、经济的体内基因转移方法.     

Homodimers of mutant tryptophan synthase alpha-subunits in Escherichia coli.

Tryptophan synthase alpha-subunit from Escherichia coli functionally exists as a heterotetramer of alpha(2)beta(2) with beta-subunit. While wild-type and mutant (F139W, T24M/F139W, and T24L/F139W) alpha-subunits were expressed as a monomer from recombinant plasmids in Escherichia coli, T24A/F139W, T24S/F139W, and T24K/F139W mutant alpha-subunits were abnormally expressed as soluble homodimers in addition to monomers. Monomers of dimer-forming mutant alpha-subunits retain high affinity to beta-subunit, high activity in stimulating catalytic activities of beta-subunit, and nearly intact content of secondary structure, indicating that the global structures of these monomers are identical to that of F139W alpha-subunit. However, fluorescence spectra of Trp139 and ANS binding indicate that significant perturbations occur in the mutant proteins. Interestingly, these defective properties of monomers caused by residue replacement were partially repaired by the dimer formation. As a result, it is suggested that dimers may be formed by domain or loop swapping, and that residue 24 may play important role in maintaining on-pathway of alpha-subunit folding.