Safety and efficacy of in vivo gene transfer into the porcine heart with replication-deficient, recombinant adenovirus vectors.

Gene transfer with replication-deficient recombinant adenovirus (Ad) vectors may provide a novel approach to the treatment of some cardiac disorders. The relative efficiency of intramyocardial vs intracoronary Ad vector injection in transducing myocardial cells remains to be determined. Further, Ad vectors are associated with localized inflammation, and this could be associated with clinically significant side-effects. Female minipigs underwent open chest surgery and the Ad vector AdCMV.NLS beta-gal was injected into the circumflex coronary artery (IC; 2 x 10(10) p.f.u.; n = 5) or the posterobasal wall of the left ventricle (i.m.; 5 x 10(9) p.f.u., n = 4; 2 x 10(10) p.f.u., n = 18). The minipigs were killed after 2-31 days and the hearts examined for evidence of beta-galactosidase activity. Minipigs underwent epicardial echocardiography immediately before, within 15 min following the i.m. injection of AdCMV.NLS beta-gal and again at the time of death. Blood samples for white blood cell count, alkaline phosphatase, total bilirubin, blood urea nitrogen, creatinine and electrolytes were obtained before i.m. and i.c. injection of the Ad vector and before death. Intramuscular injection of the Ad vector was more efficient than i.c. infusion in infecting cells in a localized area of the heart. Myocardial beta-gal activity peaked at 3-6 days after i.m. injection and returned to its control value within 1 month. Although inflammatory cells were present at the injection site, echocardiograms did not show any evidence of either segmental or global left ventricular dysfunction. No minipigs died and all blood tests remained within normal limits following either i.m. or i.c. exposure to the Ad vector. In summary, direct i.m. administration of replication-deficient, recombinant Ad vectors provides a safe and effective approach for short-term gene transfer into the heart of large mammals.

Basic fibroblast growth factor radioprotects bone marrow and not RIF1 tumor.

We examined the potential of bFGF to function as a radioprotector of bone marrow (BM). Total intravenous doses of bFGF ranged from 1 to 24 micrograms/mouse, in 2 divided doses. Whole body radiation (WBI) was given in a single fraction to C3H mice. Histologic observations were performed on femur BM at various times after bFGF (or placebo) treatment. Thigh radiation in thigh-tumor bearing mice was delivered in a single fraction. bFGF increased the LD50/30 of mice in a dose dependent fashion, with an apparent maximum protection obtained with > or = 6 micrograms given half 24 h and half 4 h before irradiation. BM histology shows prominent recovery of megakaryocytes and all cell lineages along with less loss in cellularity compared to control irradiated animals. No radioprotection of RIF1 tumors after bFGF was detected. These results indicate that bFGF may be a selective radioprotector of normal tissue.

Vigilin is a cytoplasmic protein. A study on its expression in primary cells and in established cell lines of different species.

A fusion protein composed of about two vigilin domains and beta-galactosidase was used to raise polyclonal antibodies which were affinity-purified and employed for immunoblotting and immunohistochemistry. A protein of an apparent molecular mass of 155 kDa could be stained in extracts of a variety of cells from different species and organs. Immunohistological studies on single cells showed that vigilin is accumulated in the cytoplasm. During in vitro maintenance of primary cell cultures, as well as of a growth-factor-dependent cell line, vigilin expression decreases and ceases in senescent cells. In contrast, vigilin is constitutively expressed in all other transformed cell lines of various origin studies so far. Vigilin expression can be induced in peripheral blood lymphocytes by mitogen stimulation. These observations suggest an involvement of vigilin in processes of cell activation. Immunoblot experiments demonstrating the presence of vigilin in a broad range of eukaryotes, indicate a high degree of evolutionary conservation.

Chicken vigilin gene organization and expression pattern. The domain structure of the protein is reflected by the exon structure.

Chicken vigilin was identified as a member of an evolutionary-conserved protein family with a unique repetitive domain structure. 14 tandemly repeated domains are found in chicken vigilin, all of which consist of a conserved sequence motif (subdomain A) and a potential alpha-helical region (subdomain B) [1]. We have established the physical structure of the chicken vigilin gene by restriction-fragment analysis and DNA sequencing of overlapping clones isolated from a phage lambda genomic DNA library. The chicken vigilin gene is a single-copy gene with a total of 27 exons which are distributed over a region of some 22 kbp. Exon 1 codes for a portion of the 5' untranslated region, exon 2 contains the translation start point and forms, along with exons 3 and 4, the N-terminal non-domain region. Exons 5-25 encode the vigilin domains 1-14 and the remaining exons 26 and 27 contain the non-domain C-terminal as well as the untranslated regions. The domain structure of the protein is reflected in the positioning of introns which demarcate individual domains. While domains 1-3 and 8-10 are each encoded by a single exon (5-7, 16-18); all other domains are contained in a set of two exons which are separated by introns interspersed at variable positions of the DNA segment coding for the conserved sequence motif. In conclusion, the data presented suggest that the chicken vigilin gene evolved by amplification of a primordial exon unit coding for the fundamental bipartite vigilin domain.

Complete cDNA sequence of chicken vigilin, a novel protein with amplified and evolutionary conserved domains.

The complete cDNA (4375 bp), coding for a new protein called vigilin, was isolated from chicken chondrocytes. The cDNA shows an open reading frame of 1270 amino acids which are organized in 14 tandemly repeated homologous domains. Each domain consists of two subdomains, one with a conserved sequence motif of 35 amino acids (subdomain A) and another one with a presumptive alpha-helical structure of 21-33 amino acids (subdomain B). 149 amino acids at the N-terminus and 71 amino acids at the C-terminus of vigilin do not show the characteristic domain structure. No sequence characteristic of a signal peptide has been found, which argues for an intracellular localisation of vigilin. Vigilin is highly expressed in freshly isolated chicken chondrocytes but little in chondrocytes after prolonged time in culture. Vigilin mRNA exists in two size species, 4.4 kb and 6.5 kb in length due to the usage of different polyadenylation sites. Comparison of the vigilin sequence with data bases showed a remarkable similarity to protein HX from Saccharomyces cerevisiae [Delahodde, A., Becam, A. M., Perea, J. & Jacq, C. (1986) Nucleic Acids Res. 14, 9213-9214]. The yeast protein consists of eight homologous domains with 11 conserved amino acid residues within a set of 35 amino acids. The N-terminal and C-terminal regions of vigilin and protein HX do not reveal any sequence similarity. These results, together with the demonstration of the characteristic vigilin sequence motif in a human cDNA clone, suggest that the repeats represent evolutionary conserved autonomous domains within a family of proteins found in yeast, chicken and man.