Suppression of Nonsense Mutations in the Dystrophin Gene by a Suppressor tRNA Gene

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10–15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing -galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.     

The BCL-xL and ACR-1 genes promote differentiation and reduce apoptosis in muscle fibers of mdx mice

The effects of the human BCL-xL and ACR-1 genes on dystrophin expression in cross-striated muscle fibers (CSMF) and on CSMF viability were studied in mdx mice after ballistic cotransfection with the human dystrophin minigene. In control mice, the proportion of dystrophin-positive (D(+)) and dying CSMF were 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. Introduction of the dystrophin minigene (20 micrograms of the pSG5dys plasmid) increased the proportions of D(+) and dying CSMF to 5.6 +/- 1.4% and 4.5 +/- 0.9%, respectively. When pSG5dys was introduced along with the pSFFV-Neo plasmid carrying the BCL-xL gene (10 micrograms of each plasmid per shot), the death of CSMF decreased to 3.7 +/- 1% and the proportion of D(+) CSMF significantly (P < 0.05) increased to 12.2 +/- 2.2%. Contransfection with the dystrophin minigene and the BCL-xL gene at 20 micrograms of each plasmid per shot did not stimulate generation of D(+) CSMF, but did reduce the CSMF death to 1.5 +/- 0.3%. Introduction of pSG5dys along with the pRc-CMV-10.1 plasmid containing the ACR-1 gene (10 micrograms of each plasmid per shot) reduced the proportion of D(+) CSMF to 1.1 +/- 0.5% and significantly reduced the proportion of dying CSMF to 0.9 +/- 0.3% as compared with the proportions observed in intact mice or in mice subjected to transfection with pSG5dys. Introduction of the pSG5dys plasmid substantially reduced the proportion of CSMF with peripheral nuclei, suggesting disturbed CSMF differentiation. After cotransfection with the human-dystrophin minigene, the BCL-xL and ACR-1 genes did not affect the extent of CSMF differentiation as compared with that observed in the case of the dystrophin minigene alone. Thus, ballistic transfection of mdx mice with the human dystrophin gene used along with the BCL-xL or ACR-1 gene was shown to suppress the death of muscle fibers and to expedite dystrophin synthesis and cell differentiation.     

Radioprotective effect of hypoxic hypoxia (8% O2) for different morphological elements of mouse kidney

The breathing of gas mixtures containing 8-9% O2 during irradiation of tumors has been tested at several cancer clinics (in Russia and abroad) with the purpose of decreasing the morbidity of normal issues, thus providing the possibility to increase the dose of radiation. Previous experiments have demonstrated a broad spectrum of dose modification factors (DMF) for different normal tissues as well as for different transplanted tumors, with in general larger protection of normal tissues. The present study was designed to assess the radioprotective effect for mouse kidney of breathing a gas mixture containing 8% O2 by morphometry of histological specimens. Both kidneys were locally irradiated using single fractions (11-19 Gy in air and 13-19 Gy in hypoxia) or 5 fractions separated by 24 h intervals (25-35 Gy in air and 30-40 Gy in hypoxia). Histological examination was performed 8 and 10 months after treatment. The DMF for glomeruli damage (glomerulosclerosis, ecstatic capillaries, hemorrhage) was in the range 1.25-1.29. Tubular damage showed a DMF of 1.28-1.37. Using the endpoint of development of interstitial tissue in the cortex a DMF of 1.32-1.37 was found after a single treatment, and 1.48 after fractionated irradiation. The radioprotective effect for arteriolar lesions was lower than measured using the above endpoints, namely 1.13-1.15 after single and 1.16-1.18 after fractionated irradiation. It was shown previously on groups of animals treated in the same manner that the DMF was between 1.24-1.26 when renal damage was assessed by hematocrit measurements, between 1.32-1.28 when it was evaluated by urination frequency, and 1.23-1.27 when kidney wet and dry weights were used as end-points. All these data witness that breathing 8% oxygen increases the tolerance of kidney function with a DMF above 1.2. The impact of low protection of arterioli on renal function in the late period after radiotherapy needs additional study.     

Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation

The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.     

Influence of Electroporation Conditions on Transfection of Muscle Fibers in Vivo

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electoporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Increased resistance of the radiosensitive M10 mutant cells of the L5178Y mouse lymphoma cell line to heat-induced apoptosis.

M10 cells, which are deficient in the repair of DNA DSBs and are therefore radiosensitive, are about twofold more thermoresistant than their parental L5178Y cells. We found that, after heat shock at 43 degrees C under conditions resulting in 10% survival (D(10)), M10 cells did not undergo apoptosis, whereas L5178Y cells did undergo apoptosis. M10 cells, but not L5178Y cells, constitutively expressed Hsp72 protein. Moreover, the M10 cells accumulated higher amounts of the heat-inducible form of Hsp72. The patterns of activation of the DNA-binding activity of HSF (heat-shock factor) differed in M10 and L5178Y cells. In response to heat shock, M10 cells accumulated greater amounts of Trp53 protein (formerly known as p53) than the parental cells. Cdkn1a (formerly known as p21, Waf1) was constitutively expressed and further accumulated after heat shock only in M10 cells. We suggest that heat-inducible Hsp72 to a larger extent, and constitutive Hsp72 to a lesser extent, together with Cdkn1a may be involved in the protection of M10 cells against heat-induced apoptosis. Apoptosis in these cells is likely to occur in Trp53-dependent manner.