Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Modifier”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources
PubMed1985

Endothelial cell-derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein.

Cultured rabbit and bovine aortic endothelial cells generated chemotactic activity for mouse resident peritoneal macrophages, demonstrable in the conditioned medium. This chemotactic activity was heat stable and was not extracted into chloroform/methanol. It was inhibited by addition of endothelial cell-modified low density lipoprotein (EC-modified LDL), a form of LDL shown previously to contain peroxidized lipids, increased lysophosphatidylcholine, and partially degraded apoprotein B. The chemotactic activity was also inhibited by LDL previously oxidized in the absence of cells with 5 microM Cu2+. Inhibitory activity was present in the lipid extract of EC-modified LDL but not in that of native LDL, presumably representing peroxidized lipid components. EC-modified LDL also inhibited the chemotactic activity of zymosan-activated serum. Because EC-modified LDL is taken up in part by way of the acetyl-LDL receptor, the effects of acetyl-LDL were tested. Rather than inhibiting chemotaxis, acetyl LDL showed intrinsic positive chemotactic activity as did also fucoidin and polyinosinic acid, both of which also interact with the acetyl-LDL receptor. These studies suggest mechanisms by which macrophages may be recruited into the subendothelial space by endothelial cell-derived chemotactic factors or by natural polyanions structurally related to fucoidin or polyinosinic acid and then become "trapped" there because of the inhibitory effects of peroxidized lipid components in modified forms of LDL.

Animals↗
PubMed1987

Monoclonal antibodies to DNA modified with cis- or trans-diamminedichloroplatinum(II).

Murine monoclonal antibodies that bind selectively to adducts formed on DNA by the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, or to the chemotherapeutically inactive trans isomer trans-DDP were elicited by immunization with calf thymus DNA modified with either cis- or trans-DDP at ratios of bound platinum per nucleotide, (D/N)b, of 0.06-0.08. The binding of two monoclonal antibodies to cis-DDP-modified DNA was competitively inhibited (50% control) in an enzyme-linked immunosorbent assay (ELISA) by 4-6 nM concentrations (600-900 fmol) of cis-DDP bound to DNA, (D/N)b = 0.031. Similar concentrations (4-6 nM) of cis-DDP-modified poly(dG).poly(dC) also inhibited antibody binding, whereas higher concentrations (17-36 nM) of cis-DDP-modified poly[d(AG)].poly[d(TC)] were required for inhibition. Adducts formed by cis-DDP on other synthetic DNA polymers did not inhibit antibody binding to cis-DDP-DNA. The biologically active compounds [Pt(en)Cl2], [Pt(dach)Cl2], and [Pt(NH3)2(cbdca)] (carboplatin) (where en is ethylenediamine, dach is 1,2-diaminocyclohexane, and cbdca is cyclobutane-1,1-dicarboxylate) all formed antibody-detectable adducts on DNA, whereas the inactive platinum complexes trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine) did not. The monoclonal antibodies therefore recognize a bifunctional Pt-DNA adduct with cis stereochemistry in which platinum is coordinated by two adjacent guanines or, to a lesser degree, by adjacent adenine and guanine. A monoclonal antibody raised against trans-DDP-DNA was competitively inhibited in an ELISA by 40 nM trans-DDP bound to DNA, (D/N)b = 0.022. This antibody crossreacted with unmodified, denatured DNA. Its binding to trans-DDP-DNA was selectively inhibited by trans-DDP-modified poly[d(GT)].poly[d(CA)] (50% inhibition at 1 nM bound trans-DDP). The recognition of cis- or trans-DDP-modified DNAs by monoclonal antibodies thus parallels the known modes of DNA binding of these compounds and may correlate with their biological activities.

Animals↗
PubMed1989

Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II)

DNA modified by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) was used to identify a factor in mammalian cells that binds to cis-DDP-damaged DNA and hence may play a role in repair. This factor selectively recognizes double-stranded DNA fragments modified by cis-DDP or [Pt(en)Cl2] (en, ethylenediamine). Little or no binding occurs to unmodified double-stranded DNA or to DNA modified with the clinically ineffective compounds trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine). Low levels of binding to single-stranded DNA modified by cis-DDP are observed. The apparent molecular mass of the factor in a variety of mammalian cells is approximately 100 kDa, as determined by modified Western blotting. Two recombinant phage have been isolated from a human B-cell lambda gt11 library by using a cis-DDP-modified DNA restriction fragment as a probe. The two clones have insert sizes of 1.88 and 1.44 kilobases and are aligned at their 5' ends. The polypeptides encoded by the recombinant phage exhibit DNA binding properties similar to those of the cellular factor identified in crude extracts prepared from mammalian cells. Northern analysis with one of the clones revealed an mRNA of 2.8 kilobases that is conserved in humans and rodents. The methods used here should be applicable in studies of other damage-specific DNA binding proteins.

Bacteriophage lambda↗
PubMed1994

Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces proximal tubular necrosis, a consequence of lipid peroxidation, that finally leads to a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation as monitored by formation of thiobarbituric acid-reactive substances and free 4-hydroxy-2-nonenal (HNE) was observed in the kidney homogenates of rats treated with Fe-NTA. Based on the fact that HNE is capable of reacting with cellular proteins, we attempted to detect the localization of HNE-modified proteins in rat kidney tissues with an immunohistochemical procedure. By means of an immunohistochemical technique using polyclonal antibody against the HNE-modified proteins, it was shown that HNE-modified proteins are formed in the target cells of this carcinogenesis model. HNE-modified proteins were detected in the renal proximal tubules 1 hr after i.p. administration of Fe-NTA (15 mg of iron per kg). Intense positivity was found in the cells with degeneration. After 6 hr, the level of HNE-protein conjugates decreased due to the subsequent necrosis. The intensity of the immunochemical reaction with HNE-modified proteins increased in parallel with an increase in the amounts of thiobartituric acid-reactive substances and free HNE that were found. Furthermore, histochemical detection of aldehydes by cold Schiff's reagent demonstrated that location of aldehydes was identical to that of the HNE-modified proteins determined by immunohistochemical procedures. It would thus appear that the production of HNE, a genotoxic and mutagenic aldehyde, and its reaction with proteins may play a role in Fe-NTA-induced renal carcinogenesis.

Aldehydes↗
PubMed1996

Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.

Animals↗
PubMed2001

Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase.

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.

Antioxidants↗
PubMed2001

MutS preferentially recognizes cisplatin- over oxaliplatin-modified DNA.

Loss of mismatch repair leads to tumor resistance by desensitizing cells to specific DNA-damaging agents, including the anticancer drug cisplatin. Cisplatin analogs with a diamminocyclohexane (DACH) carrier ligand, such as oxaliplatin and Pt(DACH)Cl(2), do not elicit resistance in mismatch repair-deficient cells and therefore present promising therapeutic agents. This study compared the interactions of the purified Escherichia coli mismatch repair protein MutS with DNA modified to contain cisplatin and DACH adducts. MutS recognized the cisplatin-modified DNA with 2-fold higher affinity in comparison to the DACH-modified DNA. ADP stimulated the binding of MutS to cisplatin-modified DNA, whereas it had no effect on the MutS interaction with DNA modified by DACH or EN adducts. In parallel cytotoxicity experiments, methylation-deficient E. coli dam mutants were 2-fold more sensitive to cisplatin than DACH compounds. A panel of recombination-deficient mutants showed striking sensitivity to both compounds, indicating that both types of adducts are strong replication blocks. The differential affinity of MutS for DNA modified with the different platinum analogs could provide the molecular basis for the distinctive cellular responses to cisplatin and oxaliplatin.

Adenosine Triphosphatases↗
PubMed2002

Identification in human atherosclerotic lesions of GA-pyridine, a novel structure derived from glycolaldehyde-modified proteins.

Glycolaldehyde (GA) is formed from serine by action of myeloperoxidase and reacts with proteins to form several products. Prominent among them is N(epsilon)-(carboxymethyl)lysine (CML), which is also known as one of the advanced glycation end products. Because CML is formed from a wide range of precursors, we have attempted to identify unique structures characteristic of the reaction of GA with protein. To this end, monoclonal (GA5 and 1A12) and polyclonal (non-CML-GA) antibodies specific for GA-modified proteins were prepared. These antibodies specifically reacted with GA-modified and with hypochlorous acid-modified BSA, but not with BSA modified by other aldehydes, indicating that the epitope of these antibodies could be a specific marker for myeloperoxidase-induced protein modification. By HPLC purification from GA-modified N(alpha)-(carbobenzyloxy)-l-lysine, GA5-reactive compound was isolated, and its chemical structure was characterized as 3-hydroxy-4-hydroxymethyl-1-(5-amino-5-carboxypentyl) pyridinium cation. This compound named as GA-pyridine was recognized both by 1A12 and non-CML-GA, indicating that GA-pyridine is an important antigenic structure in GA-modified proteins. Immunohistochemical studies with GA5 demonstrated the accumulation of GA-pyridine in the cytoplasm of foam cells and extracellularly in the central region of atheroma in human atherosclerotic lesions. These results suggest that myeloperoxidase-mediated protein modification via GA may contribute to atherogenesis.

Acetaldehyde↗
PubMed2002

Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the Abruptex region.

Fringe plays a key role in the specification of boundaries during development by modulating the ability of Notch ligands to activate Notch receptors. Fringe is a fucose-specific beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose moieties on the epidermal growth factor-like (EGF) repeats of Notch. To investigate how the change in sugar structure caused by Fringe modulates Notch activity, we have analyzed the sites of O-fucose and Fringe modification on mouse Notch1. The extracellular domain of Notch1 has 36 tandem EGF repeats, many of which are predicted to be modified with O-fucose. We recently proposed a broadened consensus sequence for O-fucose, C(2)X(3-5)(S/T)C(3) (where C(2) and C(3) represent the second and third conserved cysteines), significantly expanding the potential number of modification sites on Notch. Here we demonstrate that sites predicted using this broader consensus sequence are modified with O-fucose on mouse Notch1, and we present evidence suggesting that the consensus can be further refined to C(2)X(4-5)(S/T)C(3). In particular, we demonstrate that EGF 12, a portion of the ligand-binding site, is modified with O-fucose and that this site is evolutionarily conserved. We also show that endogenous Fringe proteins in Chinese hamster ovary cells (Lunatic fringe and Radical fringe) as well as exogenous Manic fringe modify O-fucose on many but not all EGF repeats of mouse Notch1. These findings suggest that the Fringes show a preference for O-fucose on some EGF repeats relative to others. This specificity appears to be encoded within the amino acid sequence of the individual EGF repeats. Interestingly, our results reveal that Manic fringe modifies O-fucose both at the ligand-binding site (EGF 12) and in the Abruptex region. These findings provide insight into potential mechanisms by which Fringe action on Notch receptors may influence both the affinity of Notch-ligand binding and cell-autonomous inhibition of Notch signaling by ligand.

Amino Acid Sequence↗
PubMed2004

Chain termination and inhibition of Saccharomyces cerevisiae poly(A) polymerase by C-8-modified ATP analogs.

The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) toward various C-2- and C-8-modified ATP analogs was examined. 32P-Radiolabeled RNA oligonucleotide primers were incubated with yPAP in the absence of ATP to assay polyadenylation using unnatural ATP substrates. The C-2-modified ATP analogs 2-amino-ATP and 2-chloro (Cl)-ATP were excellent substrates for yPAP. 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP all produced chain termination of polyadenylation, and no primer extension was observed with the C-8-halogenated derivatives 8-Br-ATP and 8-Cl-ATP. The effects of modified ATP analogs on ATP-dependent poly(A) tail synthesis by yPAP were also examined. Whereas C-2 substitution (2-amino-ATP and 2-Cl-ATP) had little effect on poly(A) tail length, C-8 substitution produced moderate (8-amino-ATP, 8-azido-ATP, and 8-aza-ATP) to substantial (8-Br-ATP and 8-Cl-ATP) reduction in poly(A) tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA primers, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was prepared. Polyadenylation of this modified RNA primer by yPAP in the presence of ATP was blocked completely. To probe potential mechanisms of inhibition, two-dimensional NMR spectroscopy experiments were used to examine the conformation of two C-8-modified AMP nucleotides, 8-Cl-AMP and 8-amino-AMP. C-8 substitution in adenosine analogs shifted the ribose sugar pucker equilibrium to favor the DNA-like C-2'-endo form over the C-3'-endo (RNA-like) conformation, which suggests a potential mechanism for polyadenylation inhibition and chain termination. Base-modified ATP analogs may exert their biological effects through polyadenylation inhibition and thus may provide useful tools for investigating polyadenylation biochemistry within cells.

Adenosine↗
PubMed2002

Explicit solvent molecular dynamics simulation of duplex formed by the modified oligonucleotide with alternating phosphate/phosphonate internucleoside linkages and its natural counterpart.

Impact of the internucleoside linkage modification by inserting a methylene group on the ability of the modified oligonucleotide to hybridize with a natural DNA strand was studied by fully solvated molecular dynamics (MD) simulations. Three undecamer complexes were analyzed: natural dT(11).dA(11) duplex as a reference and two its analogs with alternating modified and natural linkages in the deoxyadenosine chain. The isopolar, non-isosteric modified linkages were of 5'-O-PO(2)-CH(2)-O-3' (5'PC3') or 5'-O-CH(2)-PO(2)-O-3' (5'CP3') type. Simulations were performed by using the AMBER 5.0 software package with the force field completed by a set of parameters needed to model the modified segments. Both modifications were found to lead to double helical complexes, in which the thymidine strand as well as deoxyriboses and unmodified linkages in the adenosine strand adopted conformations typical for the B-type structure. For each of the two conformational richer modified linkages two stable conformations were found at 300 K: the -ggt and ggt for the 5'PC3' and ggg, tgg for the 5'CP3', respectively. Both modified chains adopted helical conformations with heightened values of the inclination parameter but without affecting the Watson-Crick hydrogen bonds.

Computer Simulation↗
PubMed2001

Malabsorption of modified food starch (acetylated distarch phosphate) in normal infants and in 8-24-month-old toddlers with non-specific diarrhea, as influenced by sorbitol and fructose.

UNLABELLED: Acetylated distarch phosphate (ADiSP) is a modified starch used in some baby foods. The bioavailability of ADiSP and a native (unmodified) starch was evaluated in 20 normal infants and 21 toddlers aged 8-24 mo with chronic non-specific diarrhea. Formulae contained 8% native or 8% modified waxy maize starch. No infant or toddler consuming Formula N (native starch) had elevated peak breath hydrogen levels (20 ppm or greater), stools clinically positive for reducing substances (0.75% or greater) or loose stools. Fourteen infants received formula M (modified starch): 2 had elevated breath hydrogen, 1 had positive stools and another had loose stools. Of the 21 toddlers fed formula M, 2 had elevated breath hydrogen, but none had positive stools or loose stools. Formula NS (native starch with 2% sorbitol) had little effect on breath hydrogen in the infants but significantly increased it in the toddlers. Formula NS produced loose stools in 2 toddlers but no clinically positive stools in any infant or toddler. Formula MS (modified starch with 2% sorbitol) elevated breath hydrogen in 3 infants and 8 toddlers, and produced positive stools in 2 infants and 2 toddlers, and loose stools in 4 infants and 7 toddlers. Formula MSF (modified starch with 2% sorbitol and 5% fructose) elevated breath hydrogen in 7 infants and 10 toddlers, positive stools in 7 infants and 6 toddlers, and loose stools or diarrhea in 7 infants and 11 toddlers. CONCLUSION: ADiSP modified starch can increase breath hydrogen and produce loose stools. Sorbitol and fructose aggravate the malabsorption, in some cases leading to frank diarrhea.

Acetylation↗
PubMed1977

Cytotoxic T cells distinguish between trinitrophenyl- and dinitrophenyl-modified syngeneic cells.

Spleen cells sensitized against trinitrophenyl (TNP)-modified stimulator cells displayed a cytotoxic effect against syngeneic TNP-modified but not dinitrophenyl (DNP)-modified target cells. The same finding was observed in the opposite direction; that is, effector cells sensitized against DNP-modified stimulator cells did not cross kill TNP-modified targets. The specificity of the anti-TNP effector cells was confirmed in a cold target competition assay. Presensitization in vivo with hapten-modified cells followed by rechallenge and testing in vitro did not alter the specificity of the response between the haptens. These data indicate that the receptor(s) on the cytotoxic T cell can distinguish between two closely related haptenic molecules.

Animals↗
PubMed1986

Voltage-dependent gating of veratridine-modified Na channels.

Na channels of frog muscle fibers treated with 100 microM veratridine became transiently modified after a train of repetitive depolarizations. They open and close reversibly with a gating process whose midpoint lies 93 mV more negative than the midpoint of normal activation gating and whose time course shows no appreciable delay in the opening or closing kinetics but still requires more than two kinetic states. Like normal activation, the voltage dependence of the modified gating can be shifted by changing the bathing Ca2+ concentration. The instantaneous current-voltage relation of veratridine-modified channels is curved at potentials negative to -90 mV, as if external Ca ions produced a voltage-dependent block but also permeated. Modified channels probably carry less current than normal ones. When the concentration of veratridine is varied between 5 and 100 microM, the initial rate of modification during a pulse train is directly proportional to the concentration, while the rate of recovery from modification after the train is unaffected. These are the properties expected if drug binding and modification of channels can be equated. Hyperpolarizations that close modified channels slow unbinding. Allethrin and DDT also modify channels. They bind and unbind far faster than veratridine does, and their binding requires open channels.

Aconitine↗
PubMed1996

Improved tumor localization and radioimaging with chemically modified monoclonal antibodies.

A method for the chemical modification of monoclonal antibodies using the heterobifunctional crosslinker succinimidyl 3-(2-pyridyldithio)propionate (SPDP), has been developed which dramatically alters the physiochemical properties of antibody reagents. For these studies, three murine monoclonal antibodies, B72.3, Lym-1, and TNT-1 were used to demonstrate the effects of chemical modification on clearance and biodistribution in tumor-bearing nude mice. In vitro, all three antibodies, modified to the same degree with SPDP, showed equal immunoreactivities and lower non-specific binding. Modified antibodies also were found to have lower isoelectric points compared to unmodified controls. In vivo, modified antibodies unexpectedly were found to have 2-6 times faster clearance in tumor-bearing nude mice similar to rates obtained with their F(ab')2 fragments. Paired-label in vivo biodistribution and external imaging experiments with intact antibodies and F(ab')2 fragments demonstrated that chemically modified antibodies gave 1.5-3 fold higher tumor uptake and retained less activity in normal organs thus markedly increasing the tumor to normal organ ratios. Because of these results, chemically modified antibodies produced clearer images at earlier time points by external scintigraphy. As "stealth" molecules, chemically modified monoclonal antibodies appear to have significantly improved uptake in tumors and faster clearance times compared to native molecules. These results suggest that alteration of the physicochemical properties of monoclonal antibodies may generate improved reagents for in vivo use.

Animals↗
PubMed1998

Control of expression and methylation of a hepatitis B virus transgene by strain-specific modifiers.

In transgenic animals, genotype-specific modifiers exert a control over transgene methylation and expression that may or may not be position dependent. These factors belong to different classes, some of them possibly related to modifiers of position-effect variegation in Drosophila. The study of hepatitis B virus (HBV) gene expression in transgenic mice has revealed the existence of many factors influencing transcription, including hormones and tissue-specific transcription factors. We now report the effect of genotype-specific modifiers on HBV surface antigen (HBsAg) expression and transgene methylation. Compared with the C57BL/6 background, the DBA/2 and 129sv backgrounds cause enhancement of HBsAg expression, with little or not effect on transgene methylation or transcription. In contrast, a single cross with a BALB/c mouse is responsible for de novo methylation and silencing of the transgene in all offspring. Several modifiers appear to segregate in the progeny of a transgenic E36 male mouse crossed with (C57BL/6 x BALB/c) F1 females, with the emergence of a high-expressor group. Our observations suggest that different modifiers act cooperatively, at both the transcriptional and post-transcriptional levels, as part of a complex system regulating transgene expression. This transgenic model provides a system to genetically map new mouse strain-specific modifiers, some of them involved in epigenetic modification and transcription control.

Animals↗
PubMed1997

Use of cloned genetically modified human fibroblasts to assess long-term survival in vivo.

Because human fibroblasts are easily brought to tissue culture conditions and can be stably transduced with retroviral vectors encoding transgenes ex vivo, genetically modified fibroblasts are frequently considered in strategies to correct disease with gene therapy. This enthusiasm has been dampened by studies showing that transgene expression by genetically modified fibroblasts diminishes with time in vivo, but not in vitro, for reasons that are unclear. We elected to study this problem using cloned human fibroblasts that had been cloned by limiting dilution and stably transduced with a retroviral vector encoding lacZ ex vivo. These were seeded onto a nonbiodegradable nylon matrix that was transplanted to nude mice. Transgene expression was followed prospectively by histologic exam. Data show that human fibroblasts can withstand the pressure of cloning by limiting dilution. In addition, they can be passaged from 10 to > 20 times, and > 1 x 10(20) of genetically modified fibroblasts can be generated as progeny of one cell. Loss of transgene expression by the cloned genetically modified fibroblasts in vivo occurs in an orderly and progressive fashion, but is not complete by 4 months. Neither the loss nor the persistence of expression appear to be random. These observations are most compatible with the thesis that a major cause of the loss of transgene expression in vivo is secondary to apoptosis of the genetically modified fibroblast. Loss of expression of transgenes in senescent genetically modified fibroblasts occurs more rapidly than in their presenescent counterparts in the age-neutral, in vivo setting of the nude mouse.

Animals↗
PubMed2004

Comparison of the efficiency of transduction of leukemic cells by fiber-modified adenoviruses.

Efficient gene transfer with adenoviral type 5 (Ad5) vectors depends on the initial attachment of their fiber, which binds the coxsackie-adenovirus receptor (CAR), and their subsequent internalization, mediated by the interaction of viral penton base with target cell alphav integrins. We previously demonstrated that human leukemic cells lack these receptors and are therefore resistant to Ad5 transduction, limiting efforts to genetically modify these cells. Human leukemic blasts are, however, susceptible to transduction with an adenovector made CAR independent by substitution of a chimeric Ad5/35 fiber [Yotnda et al. (2001). Gene Ther. 8, 930-937]. Other receptors can also be targeted with recombinant ligand moieties incorporated into adenovirus fiber. We have determined which of these fiber-modified adenovectors is most effective at modifying human primary leukemia cells, and lines. We used a replication-incompetent Ad5-beta-gal vector, in which the Ad5 fiber was replaced with fiber from various adenovirus serotypes (Ad35 and Ad11), or modified either with variable length polylysine (K4, K7, K21) or RGD-4C peptide. All the modified fiber vectors transduced primary leukemia cells and cell lines more efficiently than Ad5. Polylysine-substituted Ad5F/K21 and peptide-modified Ad5F/RGD vectors were most effective overall (up to 100% efficiency), whereas Ad5F/RGD was the most effective at transducing B cell acute lymphoblastic leukemia cells (90% efficiency). Ad5F/K21 and Ad5F/RGD should be of value for the genetic modification of human primary leukemia cells.

Adenoviridae↗