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PubMed1995

The use of umbilical cord blood as a cellular source for correction of genetic diseases affecting the hematopoietic system.

Human umbilical cord blood contains abundant primitive and committed hematopoietic progenitors and has been used as an alternative source of reconstituting hematopoietic stem cells. Recent advances in the understanding of molecular aspects of multiple diseases and improvements in technology associated with prenatal diagnosis now allow the in utero identification of many genetic diseases affecting the hematopoietic system. Advances in technology raise the potential for genetic correction and subsequent transplantation of autologous cord and placental blood hematopoietic stem cells into affected patients prior to expression of the disease phenotype. This review will summarize the recent data on advances in prenatal diagnosis, characterization of the biology of cord blood stem cells, and efforts at developing methods for genetic transduction of cord blood hematopoietic stem/progenitor cells.

Animals↗
PubMed2001

APC, signal transduction and genetic instability in colorectal cancer.

Colorectal cancer arises through a gradual series of histological changes, each of which is accompanied by a specific genetic alteration. In general, an intestinal cell needs to comply with two essential requirements to develop into a cancer: it must acquire selective advantage to allow for the initial clonal expansion, and genetic instability to allow for multiple hits in other genes that are responsible for tumour progression and malignant transformation. Inactivation of APC--the gene responsible for most cases of colorectal cancer--might fulfil both requirements.

Animals↗
PubMed2000

A Stat3-interacting protein (StIP1) regulates cytokine signal transduction.

Genetic and biochemical studies have led to the identification of the Stat3-Interacting Protein StIP1. The preferential association of StIP1 with inactive (i.e., unphosphorylated) Stat3 suggests that it may contribute to the regulation of Stat3 activation. Consistent with this possibility, StIP1 also exhibits an affinity for members of the Janus kinase family. Overexpression of the Stat3-binding domain of StIP1 blocks Stat3 activation, nuclear translocation, and Stat3-dependent induction of a reporter gene. These studies indicate that StIP1 regulates the ligand-dependent activation of Stat3, potentially by serving as a scaffold protein that promotes the interaction between Janus kinases and their Stat3 substrate. The ability of StIP1 to associate with several additional members of the signal transducer and activator of transcription family suggests that StIP1 may serve a broader role in cytokine-signaling events.

3T3 Cells↗
PubMed2004

Genetic signal transduction by nitrosyl-iron complexes in Escherichia coli.

Nitrosyl-iron complexes used as aqueous preparations of binuclear dinitrosyl-iron complex with glutathione (DNICglu), initially polycrystalline preparations of binuclear tetranitrosyl-iron complex with thiosulfate (TNICthio), and also binuclear tetranitrosyl-iron complex with aminotriazole (TNICatria) and mononuclear dinitrosyl-iron complex with triazole (DNICtria) in the concentration to 0.1 mM activated expression of the soxS and sfiA genes in Escherichia coli. Higher concentrations of polycrystalline preparations of low stability in aqueous solutions were cytotoxic, whereas DNICglu, which is more stable in water (up to two days), increased the gene expression on increase in its concentration to 0.5 mM. The iron chelating agent o-phenanthroline completely inhibited the gene expression induced by all compounds studied. The genetic signal transduction seemed to be realized not by nitric oxide molecules and/or iron ions released in solutions but directly by the complexes themselves, which activate transcriptional proteins by transfer onto them of nitrosyl-iron groups [Fe+(NO+)2].

Electron Spin Resonance Spectroscopy↗
PubMed1998

Generalized transduction for genetic linkage analysis and transfer of transposon insertions in different Staphylococcus epidermidis strains.

Staphylococcus epidermidis phage 48 was used to efficiently transduce plasmid pTV1ts and a chromosomal Tn917 insertion M27 from S. epidermidis 13-1 to biofilm-producing clinical S. epidermidis isolates 1457, 9142, and 8400. The Tn917 insertion leading to the biofilm-negative phenotype of transposon mutant M10 was sequentially transduced to biofilm-producing S. epidermidis 1457 using S. epidermidis phage 48 and then, using the resulting biofilm-negative transductant 1457-M10 as a donor, into several unrelated biofilm-producing clinical S. epidermidis isolates using S. epidermidis phage 71. All resultant transductants displayed a completely biofilm-negative phenotype. In addition, S. epidermidis phage 71 was adapted to S. epidermidis 1457 and 8400, which allowed generalized transduction of transposon insertions in these wild-type strains. As Tn917 predominantly transposed into endogenous plasmids of all three strains used, an efficient system for chromosomal transposon mutagenesis was established by curing of S. epidermidis 1457 of a single endogenous plasmid p1457 by sodium dodecylsulfate treatment. After transduction of the resulting derivative, S. epidermidis 1457c with pTV1ts, insertion of transposon Tn917 to different sites of the chromosome of S. epidermidis 1457c was observed. Biofilm-producing S. epidermidis 1457c x pTV1ts was used to isolate a biofilm-negative transposon mutant (1457c-M3) with a chromosomal insertion apparently different from two previously isolated isogenic biofilm-negative transposon mutants, M10 and M11 (Mack, D., M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs: Infect Immun 62 [1994] 3244-3253). S. epidermidis phage 71 was used to prove genetic linkage between transposon insertion and altered phenotype by generalized transduction. In combination with phage transduction, 1457c x pTV1ts will be a useful tool facilitating the study of bacterial determinants of the pathogenicity of S. epidermidis.

Blotting, Southern↗
PubMed2001

The genetics of signal transduction and the outcome of diagnostic tests in growth retardation.

The effects of 'normal' genetic variability of signal transduction on endocrine function may be more evident during stimulation tests than is observed in basal states, thereby contributing to a greater understanding of the possible role of signal transduction genetics in the pathogenesis of endocrine disorders. In the present study, we have studied the outcome of growth hormone (GH) stimulation testing by insulin in growth-retarded children in relation to the genotype of ACP1 (acid phosphatase locus 1; also referred to as cLMWPTP, cytosolic low molecular weight phosphotyrosine phosphatase). ACP1 is an enzyme, expressed as two distinct isoforms designated F and S, that down-regulates insulin receptor signal transduction and which shows a genetic polymorphism with strong quantitative enzymatic differences among genotypes. In this study, we examined 116 growth-retarded children of which 101 were genotyped for ACP1. We found that the basal level of GH is higher in ACP1 genotypes with low concentrations of the S isoform than in genotypes with high S isoform concentrations (P<0.02). Additionally, during GH stimulation with insulin, the genotypes with low S isoform concentrations were found to perform better (P<0.005) and to react more promptly than the genotypes with high S isoform concentrations (P<0.05). These findings suggest that high S isoform ACP1 activity slows down the effect of insulin, resulting in a retardation of its metabolic effect.

Acid Phosphatase↗
PubMed1975

[Factors influencing transduction of genetic determinants of penicillinase activity and pathogenicity in staphylococcus aureua. II. Antiphage activity of acridine derivatives].

Acridine dyes examined earlier (acrichine, acridine orange, proflavine and rivanol) and newly-synthesized preparations (acridines No. No. 37--40) were studied in the capacity of nonspecific agents influencing the lytic cycle in development of staphylococcus phages. Acrichine and acridine No. 37 failed to prevent lysis of the indicator staphylococcus cultures (strains 16/160 and 8325) by bacteriophages; proflavine, rivanol, acridines No. No. 39--40 produced a marked inhibitory effect; acridine orange and acridine No. 38 inhibited the staphylococcus lysis completely. Some preparations could be used to investigate the transduction phenomenon.

Acridines↗
PubMed2003

The Theodore Bücher lecture. Investigating signal transduction with genetically encoded fluorescent probes.

Ca2+ and cAMP are ubiquitous second messengers in eukaryotes and control numerous physiological responses ranging from fertilization to cell death induction. To distinguish between these different responses, their subtle regulation in time, space and amplitude is needed. Therefore, the characterization of the signalling process requires measurement of second messengers with tools of precise localization, high dynamic range and as little disturbance of cell physiology as possible. Recently, fluorescent proteins of marine jellyfish have given rise to a set of genetically encoded biosensors which fulfil these criteria and which have already led to important new insights into the subcellular handling of Ca2+ and cAMP. The use of these probes in combination with new microscopical methods such as two-photon microscopy now enables researchers to study second messenger signalling in intact tissues. In this review, the genetically encoded measurement probes and their origin are briefly introduced and some recent insights into the spatio-temporal complexity of both Ca2+ and cAMP signalling obtained with these tools are discussed.

Aequorin↗
PubMed2000

Genetically similar transduction mechanisms for touch and hearing in Drosophila.

To test the effects of mechanosensory mutations on hearing in Drosophila, we have recorded sound-evoked potentials originating from ciliated sensory neurons in Johnston's organ, the chordotonal organ that is the sensory element of the fly's antennal ear. Electrodes inserted close to the antennal nerve were used to record extracellular compound potentials evoked by near-field sound stimuli. Sound-evoked potentials are absent in atonal mutant flies, which lack Johnston's organ. Mutations in many genes involved in mechanotransduction by tactile bristles also eliminate or reduce the Johnston's organ response, indicating that related transduction mechanisms operate in each type of mechanosensory organ. In addition, the sound-evoked response is affected by two mutations that do not affect bristle mechanotransduction, beethoven (btv) and touch-insensitive-larvaB (tilB). btv shows defects in the ciliary dilation, an elaboration of the axoneme that is characteristic of chordotonal cilia. tilB, which also causes male sterility, shows structural defects in sperm flagellar axonemes. This suggests that in addition to the shared transduction mechanism, axonemal integrity and possibly ciliary motility are required for signal amplification or transduction by chordotonal sensory neurons.

Acoustic Stimulation↗
PubMed1998

Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma.

Recombinant adenovirus (Adv)-mediated gene transduction is a powerful technology for cancer gene therapy. In this article, we report the generation of a fiber-mutant Adv vector, using the Adv genomic DNA-terminal protein complex (DNA-TPC) cotransfection method. First, a fiber-mutant construct in a plasmid carrying the right-side two-thirds of the human adenovirus type 5 (Ad5) genome (pTR) was cotransfected with Ad5 DNA-TPC, yielding the recombinant Adv with the desired fiber mutation. The DNA-TPC from the mutant Adv was then utilized to produce a second-step recombinant Adv with an expression cassette in the place of E1. By this procedure, we generated a fiber mutant, F/K20, that has a linker and a stretch of 20 lysine residues added at the C terminus of the fiber. By using Adv carrying a reporter lacZ gene (AxCAZ2) with either F/K20 or wild-type fiber (F/wt), we examined the transduction efficiency of F/K20-Adv. No significant difference in the transduction efficiency between F/K20 and F/wt-Adv was observed for a human fibroblast line, WI-38, or various tumor cell lines, including melanoma, prostate, esophageal, and pancreatic cancer lines. In clear contrast, F/K20-Adv showed a remarkably enhanced efficiency in genetic transduction of human glioma cells. In all four human glioma lines tested, the multiplicities of infection (MOIs) for transduction of 50% of the population (ED50) were decreased with F/K20-Adv compared with F/wt-Adv: 7-fold for T98G, 14-fold for U251, 9-fold for U373, and 42-fold for U87 cells. Therefore, we attempted to apply F/K20-Adv for gene therapy of malignant glioma. Glioma cells infected with F/K20-Adv carrying genes for interleukin 2 or interleukin 12 produced a high level of each cytokine at a much lower MOI than did cells infected with F/wt-Adv. Infection with F/K20-Adv carrying the wild-type p53 tumor suppressor gene resulted in an enhanced level of p53 protein expression and an increased incidence of F/K20-Adv in transduction efficiency for malignant glioma, providing promising tools for gene therapy.

Adenoviridae↗
PubMed2001

Protein transduction: an alternative to genetic intervention?

Protein transduction, an emerging technology with potential applications in gene therapy, can best be described as the internalisation of proteins into the cell, from the external environment. This process relies on the inherent property of a small number of proteins and peptides of being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offers an alternative to gene therapy for the delivery of therapeutic proteins into target cells. This review describes the three most commonly used protein transduction vehicles; the antennapedia peptide, the herpes simplex virus VP22 protein and HIV TAT protein transduction domain. The future prospects for the application of this technology in gene therapy are also discussed.

Antennapedia Homeodomain Protein↗
PubMed2004

Imaging and therapy of tumors induced to express somatostatin receptor by gene transfer using radiolabeled peptides and single chain antibody constructs.

The fields of radioimmunodetection and radioimmunotherapy began with an initial paradigm that a targeting molecule (eg, antibody) carrying a radioisotope had the potential of selectively imaging and delivering a therapeutic dose of radiation to tumor sites. A second paradigm was developed in which injection of the targeting molecule was separated from injection of a short-lived radioisotope-labeled ligand (so-called "pretargeting strategy"). This strategy has improved radioisotope delivery to tumors in animal models, enhanced radioimmune imaging in man, and therapeutic trials are in an early phase. We proposed a third paradigm to achieve radioisotopic localization at tumor sites by inducing tumor cells to synthesize a membrane expressed receptor with a high affinity for infused radiolabeled ligands. The use of gene transfer technology to induce expression of high affinity membrane receptors can enhance the specificity of radioligand localization, while the use of radioisotopes with the ability to deliver radiation damage across several cell diameters will compensate for less than perfect transduction efficiency. This approach was termed "Genetic Radioisotope Targeting Strategy." Using this strategy, induction of high levels of gastrin releasing peptide receptor or human somatostatin receptor subtype 2 expression and selective tumor uptake of radiolabeled peptides was achieved. The advantages of the genetic transduction approach are (1) constitutive expression of a tumor-associated antigen/receptor is not required; (2) tumor cells are altered to express a new target receptor or increased quantities of an existing receptor at levels that may significantly improve tumor targeting of radiolabeled ligands compared with normal tissues; (3) gene transfer can be achieved by intratumoral or regional injection of gene vectors; (4) it is feasible to target adenovirus vectors to receptors overexpressed on tumor cells by modifying adenoviral tropism (binding) so that the virus will be targeted specifically to the desired tumor; and (5) it is possible to coexpress the receptor gene and a therapeutic gene, such as cytosine deaminase, for molecular prodrug therapy to produce an enhanced therapeutic effect.

Animals↗
PubMed1975

Mutations simultaneously affecting endonuclease II and exonuclease III in Escherichia coli.

We studied mutants of E. coli originally identified as being deficient in either endonuclease II (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.30) or exonuclease III [deoxyribonucleate (double-stranded) 5'-nucleotidohydrolase, EC 3.1.4.27] activity. Twelve independently derived mutants were tested, including three new endonuclease II mutants. Deficiency of one enzyme was always accompanied by deficiency of the other. Furthermore, temperature-sensitivity of one activity was always accompanied by temperature-sensitivity of the other, and the enzymes were co-purified. The results suggested a physical association between exonuclease III and endonuclease II, which may be of advantage in the excision-repair of DNA. A thermolabile endonuclease II was purified from one of the new mutants, indicating that it had an altered structural gene. This mutation, and all similar ones mapped by genetic transduction, was located between the pncA and aroD genes on the E. coli chromosome. One mutant had a prolonged generation time, an increased sensitivity to the alkylating agents methyl-methanesulfonate and mitomycin C, and a decreased plating efficiency for bacteriophage lambda, but no marked sensitivity to ultraviolet or gamma-irradiation. Its enzymatic and biological abnormalities were simultaneously revertible, suggesting they were caused by a single mutation. These results suggested a role for these enzymes in normal cell growth processes and in the repair of alkylation damage.

Chromosome Mapping↗
PubMed1992

High-level expression of recombinant human tPA in cultivated canine endothelial cells under varying conditions of retroviral gene transfer.

Successful genetic transduction of endothelial cells (EC) provides a theoretic means of increasing luminal secretion of tissue-type plasminogen activator (tPA) and lessening arterial and venous thrombotic processes. To identify the duration and number of retroviral exposures for an optimal tPA expression, enzymatically derived adult canine jugular venous EC were subjected to different exposure regimens using an amphotropic murine retroviral vector, MFG, containing the human tPA gene. Human tPA antigen secretion and its functional activity were determined at 2 days (subconfluent cells) and 14 days (confluent cells) after retroviral exposure. High-level secretion of human tPA was detected among transduced EC in all experimental groups. No secretion of human tPA occurred in control EC exposed to media alone. At 2 days after transduction, no significant differences in tPA secretion rates occurred among the different exposure regimens. At 14 days, the 12-hour X two-exposure group exhibited higher tPA secretion rates than all other exposure regimens (analysis of variance, p < 0.05). All exposure groups at 14 days exhibited significantly higher tPA secretion compared with those at 2 days (analysis of variance, p < 0.05). The presence of retroviral sequences in the genome of transduced EC was confirmed by Southern blot analysis. At 14 days, increased EC numbers were observed in vector-exposed wells compared with controls. Human tPA functional activity paralleled tPA antigen secretion. Genetically modified canine EC are capable of high levels of constitutive expression of human tPA after relatively short exposures to a retroviral vector containing the reporter gene. Increased cell number of tPA-transduced EC in culture suggests that tPA also may have other biologically important effects. These results support the efficacy of MFG-tPA gene transfer as a means of genetically modifying EC fibrinolytic activity and establishes the potential of this technology in vivo.

Animals↗