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At least 865 records · Page 48Linked to original sources
PubMed1995

The cytotoxicity of mitomycin C and adriamycin in genetically engineered V79 cell lines and freshly isolated rat hepatocytes.

The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and beta-naphthoflavone (beta NF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytotoxicity was observed in V79, SD1 and XEM2 cells. Addition of metyrapone (MP, an inhibitor of P450IIB1) and alpha-naphthoflavone (alpha NF, an inhibitor of P450IA1) had no effect on the ADR-induced cytotoxicity in SD1 and XEM2 cells, respectively. Likewise, MMC was equitoxic in V79 and SD1 cells. Co-incubation of SD1 cells with MP did not alter MMC-induced cytotoxicity. MMC, however, showed a decreased cytotoxicity in XEM2 cells when compared to the parental V79 cells. Unexpectedly, the cytotoxicity of MMC in XEM2 cells was increased by alpha NF to the same level as observed in the parental V79 cells. In contrast to V79- and V79-derived cells, in freshly isolated hepatocytes from PB or beta NF-induced rats, MMC was cytotoxic (measured as lactate dehydrogenase leakage) within 3 h of incubation. ADR, however, was only cytotoxic to the hepatocytes when intracellular glutathione was first depleted by diethylmaleate. The MMC- and ADR-induced cytotoxicity was found to be more pronounced in PB-hepatocytes than in beta NF-hepatocytes. Contrary to the findings in the V79-derived cells, MP afforded complete protection against both MMC- and ADR-induced cytotoxicity in PB-hepatocytes, whereas alpha NF only partially inhibited the cytotoxicity of MMC in beta NF-hepatocytes. In conclusion, we have demonstrated that PB-inducible P450s play a role in the cytotoxicity of both MMC and ADR in freshly isolated PB-hepatocytes but that P450IIB1 does not in genetically reconstituted SD1 cells. P450IA1, however, decreased the cytotoxicity of MMC in the XEM2 cells. The ADR-induced cytotoxicity, which was observed in XEM2 cells, was not mediated by P450IA1. The present study underscores the complexity in the comparison of ADR- and MMC-induced cytotoxicities in normal and tumor cells.

Animals↗
PubMed2006

Effects of Runx2 genetic engineering and in vitro maturation of tissue-engineered constructs on the repair of critical size bone defects.

Genetic and tissue engineering strategies are being pursued to address the clinical limitations of current bone grafting materials. Based on our previous work demonstrating that overexpression of the Runx2 osteoblastic transcription factor and in vitro construct maturation synergistically enhanced in vivo mineralization in an ectopic site (Byers et al., Tissue Eng 2004;10:1757-1766), we examined the effects of these two parameters on the repair of critical size bone defects. Primary rat bone marrow stromal cells transduced with Runx2 or control (no Runx2 insert) retroviral vector were seeded onto 3D fused deposition-modeled polycaprolactone scaffolds. Runx2-modified cells produced biologically-equivalent mineralized matrices at nearly 2-fold higher rates than control cells. Constructs cultured in vitro for 1 day (immature) or 21 days (mineralized) were subsequently implanted into critical size calvaria defects in syngeneic rats, and bone healing was analyzed by micro-CT and histomorphometry at 28 days. Runx2-modified and control constructs precultured for 1 day healed to a greater extent than defects receiving no implant. Cell-free scaffolds yielded equivalent levels of bone formation as constructs precultured for 1 day. Interestingly, defects treated with control cell-seeded constructs precultured for 21 days exhibited low bone formation compared to other construct treatments, and repair was comparable to empty defects. In contrast, Runx2-modified constructs precultured for 21 days contained twice as much bone as control constructs precultured for 21 days and equivalent levels of new bone as cell-free and 1 day precultured constructs. These results demonstrate interplay between Runx2 genetically-modified cells and in vitro construct maturation in bone healing responses.

Absorbable Implants↗
PubMed1990

Genetic engineering, isolation and characterization of a truncated Escherichia coli elongation factor Tu comprising domains 2 and 3.

A deletion mutant of a plasmid born Escherichia coli tufA gene, which codes for a truncated elongation factor Tu comprising domains 2 and 3, has been constructed by genetic engineering. This gene was overexpressed in E. coli, and a polypeptide representing the truncated elongation factor Tu was isolated, purified to near homogeneity, crystallized and characterized physico-chemically as well as biochemically. Circular dichroism spectroscopy and limited tryptic digestion demonstrate that the isolated domain pair 2 and 3 behaves like an independent folding unit which adopts a similar secondary and most likely, tertiary, structure to that present in the intact elongation factor Tu. However, the isolated domain pair 2 and 3 does not interact with aminoacyl-tRNA or the antibiotic kirromycin, two ligands which were shown previously by cross-linking experiments to be in contact with amino acid residues located in domains 1 and 2, and domain 3, respectively. The results suggest that the isolated domain pair 2 and 3 by itself forms too few contacts with these ligands to form a stable complex. Furthermore, the data suggest that domain 1 in intact EF-Tu, in a subtle but nevertheless decisive manner, alters the conformation of the other two domains in such a way that all three domains cooperatively create a high affinity binding site for aminoacyl-tRNA and the antibiotic kirromycin.

Circular Dichroism↗
PubMed1997

New insights into the ontogeny of breathing from genetically engineered mice.

Development of breathing behavior depends on the coordinated maturation of central and peripheral neural pathways, respiratory muscles, airways, and lung tissues. Each of these components contains cellular elements in which derangements of gene expression may perturb development of normal respiratory function. Application in recent years of genetic engineering techniques has led to detailed analyses of gene structure and function. In particular, targeted gene deletions provide the opportunity to relate gene function to physiologic mechanisms in intact animals. This review summarizes recent studies in mice designed to alter, by targeted disruption of specific genes, development of individual components of the respiratory control system. We also discuss an example of the human therapeutic potential of transgenic methods.

Animals↗
PubMed1996

Inhibition of viral replication by genetically engineered mutants of the duck hepatitis B virus core protein.

The hepatitis B virus (HBV) nucleocapsid consists of 240 viral core proteins that are arranged in a highly symmetrical structure, HBV replication can only take place inside intact nucleocapsids. In the present study, we investigated whether genetically engineered core mutants can inhibit viral replication by interfering with the formation of intact nucleocapsids. Using the duck hepatitis B virus (DHBV) model, a series of core protein mutants was generated. Polymerase chain reaction-amplified fragments from the bacterial lacZ gene expressing up to 282 amino acids were added either to the amino- or carboxy-terminus of the DHBV core protein. In addition, carboxy-terminal extensions were generated by fusing the DHBV core protein with the DHBV small surface protein or various fragments of the viral polymerase. Finally, the green fluorescent protein (GFP) was fused in-frame to the carboxy-terminus of the DHBV core protein. In this chimeric protein, GFP is still functional and can act as a reporter molecule. The various core protein mutants were tested for their potential antiviral activity by cotransfection with a replication-competent DHBV construct into the avian hepatoma cell line LMH. Carboxy-terminal, but not amino-terminal, DHBV core mutants inhibited DHBV replication by up to 90% at an effector-to-target ratio of 1:10, thus displaying a dominant negative phenotype. Antiviral activity was species-specific and caused by posttranslational interference with viral replication. The DHBV core-GFP fusion protein should be an ideal tool to assess the antiviral potential of dominant negative core proteins in vivo.

Amino Acid Sequence↗
PubMed2000

The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting.

NIH sponsored a meeting of medical and veterinary pathologists with mammary gland expertise in Annapolis in March 1999. Rapid development of mouse mammary models has accentuated the need for definitions of the mammary lesions in genetically engineered mice (GEM) and to assess their usefulness as models of human breast disease. The panel of nine pathologists independently reviewed material representing over 90% of the published systems. The GEM tumors were found to have: (1) phenotypes similar to those of non-GEM; (2) signature phenotypes specific to the transgene; and (3) some morphological similarities to the human disease. The current mouse mammary and human breast tumor classifications describe the majority of GEM lesions but unique morphologic lesions are found in many GEM. Since little information is available on the natural history of GEM lesions, a simple morphologic nomenclature is proposed that allows direct comparisons between models. Future progress requires rigorous application of guidelines covering pathologic examination of the mammary gland and the whole animal. Since the phenotype of the lesions is an essential component of their molecular pathology, funding agencies should adopt policies ensuring careful morphological evaluation of any funded research involving animal models. A pathologist should be part of each research team.

Animals↗
PubMed1988

Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen-combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.

Amino Acid Sequence↗