Marcel Herman Jozef Ruiters

Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system

Parameters that affect cellular transfection as accomplished by introducing DNA via carriers composed of cationic synthetic amphiphiles, have been investigated, with the aim to obtain insight into the mechanism of DNA translocation. Such insight may be exploited in optimizing carrier properties of synthetic amphiphiles for molecules other than nucleic acids. In the present work, the interaction of vesicles composed of the cationic amphiphile dioleyloxy-propyl-trimethylammonium chloride (DOTMA) with cultured cells was examined. The results show that optimal transfection is dependent on the concentration of lipid, which determines the efficiency of vesicle interaction with the target cell membrane, as well as the toxicity of the amphiphiles towards the cell. A low lipid/DNA ratio prevents the complex from interacting with the cell surface, whereas at a relatively high amphiphile concentration the complex becomes toxic. Translocation efficiency is independent of the initial vesicle size but is affected by the size of the DNA. An incubation time of the DNA/amphiphile complex and cells of approx. 2-4 h is required for obtaining efficient transfection. In conjunction with observations on DNA/amphiphile complex-induced hemolysis of erythrocytes, a mechanism of DNA-entry is proposed which involves translocation of the nucleic acids through pores across the membranes rather than delivery via fusion or endocytosis. Dioleoylphosphatidylethanolamine, a phospholipid frequently used in a mixture with DOTMA (lipofectin) strongly facilitates this pore formation. Translocation of the DNA is effectively prevented when the cells are pretreated with Ca2+ or pronase. These observations suggest that Ca(2+)-sensitive cell surface proteins play a role in amphiphile-mediated DNA translocation.

Telomere length in breast cancer patients before and after chemotherapy with or without stem cell transplantation

High-dose chemotherapy and peripheral blood stem cell transplantation (PBSCT) may accelerate telomere length loss in haematopoietic stem cells. As data including pre-and post-treatment samples are lacking, we studied leukocyte telomere length and telomerase activity before and after treatment in breast cancer patients randomized to receive 5 adjuvant courses FEC (5-FU, epirubicin and cyclophosphamide) (n= 17), or 4 × FEC followed by high-dose cyclophosphamide, thiotepa, carboplatin and autologous PBSCT (n= 16). Haemoglobin, MCV, leukocyte-and platelet numbers were assessed prior to (t0), 5 months after (t1) and 9 months after chemotherapy (t2); these parameters were decreased at t1and t2compared to t0(high-dose: all parameters; standard-dose: leukocytes and platelets), and all parameters were lower after high-dose than standard-dose treatment at t1. Paired individual leukocyte samples of t0 and t1showed telomere length change (determined by telomere restricted fragment (TRF) assay) ranging from +0.8 to –2.2 kb, with a decreased TRF length in 9 patients of both groups. Telomerase activity (determined by TRAP assay) was below detection limit in leukocyte samples of t0 and t1. Thus, standard-and high-dose chemotherapy negatively affect haematological reconstitution in this setting. In individual patients, telomere length can be remarkably changed following haematological proliferative stress after treatment. http://www.bjcancer.com

Synthesis of Pyridinium Amphiphiles Used for Transfection and Some Characteristics of Amphiphile/DNA Complex Formation

Pyridinium amphiphiles have found practical use for the delivery of DNA into cells. Starting from 4-methylpyridine, a general synthesis has been devised for the production of pyridinium amphiphiles which allows variation in both the hydrophobic part and in the headgroup area of the compounds. By means of differential scanning microcalorimetry, zeta potential, particle size measurements and cryo electron microscopy, some characteristics of the pyridinium amphiphile/ DNA complexes have been determined.

Localization of dopamine D2 receptor mRNA with non-radioactive in situ hybridization histochemistry.

A digoxigenin-labeled antisense 42-mer oligonucleotide was used for the localization of the dopamine D2 receptor mRNA in the rat brain. The digoxigenin label was identified with alkaline phosphatase conjugated sheep-anti-digoxigenin. In good analogy with the known terminal fields of the dopaminergic system, various nuclei throughout the brain were labeled. Positive in situ hybridization signals were also found in dopamine cell groups of the substantia nigra and ventral tegmental area and in regions where a dopaminergic innervation is controversial, like the cerebellar cortex and the hippocampus. The non-radioactive in situ hybridization procedure described, shows the localization of the dopamine D2 receptor mRNA with a very high contrast and an optimal histological resolution.

Differential Glutathione Peroxidase mRNA Up-Regulations in Rat Forebrain Areas after Transient Hypoxia-Ischemiaa

The biological reduction of oxygen generates reactive oxygen species that are toxic to cells. To protect themselves against such toxic moieties, cells have evolved antioxidant defence mechanisms involving enzymes and vitamines. The enzymatic protection proceeds in two steps: 1) the conversion of superoxide anions (“0;) to hydrogenperoxide (HzO,) by superoxide dismutase (SOD) and (2) the subsequent conversion of hydrogenperoxide to water by glutathione peroxidase (GPx) and/or catalase (Cat). If, however, the activity or amount of SOD is increased without concomitant increase of GPx and/or Cat, then hydrogenperoxide accumulates and reacts with transition metals in the Fenton’s reaction to produce hydroxyl radicals (“OH). These hydroxyl radicals are among the most noxious radical species known and react with macromolecules, in particular with the polyunsaturated fatty acids of membrane lipids. This process, known as lipid peroxidation, initiates in the lipid bilayers cascades of free-radical-generating reactions that disrupt the membrane integrity and allow eventually entry of calcium ions into the cells. Overflow of the redundant calcium into the mitochondria generates a cellular energy crisis that leads to cell death.’.x Early and late neuronal degenerations observed in circumscribed brain regions after focal or global cerebral ischemia may be triggered by free radicals that are

Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18((R)):DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

Spinal cord stimulation and the induction of c-fos and heat shock protein 72 in the central nervous system of rats

For more than a decade, spinal cord stimulation (SCS) has been used as an adjuvant treatment for patients who are unresponsive to conventional therapies for angina pectoris. Many studies showed that SCS has both electro‐analgesic and anti‐ischemic effects. Nonetheless, the biological substrates by which SCS acts have not yet been unraveled, although recently areas in the brain have been described that show changes in blood flow, following SCS, and during provocation of angina. In search of a putative mechanism of action of SCS, we hypothesized that SCS affects processing of nociceptive information within the central nervous system (CNS). Moreover, it may alter the limbic system activity that maintains the balance between sympathetic and parasympathetic activity in the heart. Hence, we have developed a rat model to investigate its suitability for studying the induction of neural activity during SCS. To characterize neural activity, we used the expression of both the immediate early gene c‐fos and the heat shock protein 72 (HSP72). c‐Fos was used to identify structures in the CNS affected by SCS, and HSP72 was applied in order to ascertain whether SCS might operate as a stressor.

Expression of annexin and Annexin-mRNA in rat brain under influence of steroid drugs

Brain tissue of rats pretreated with methylprednisolone or with the 21-aminosteroid U74389F, and that of untreated control rats, was assessed for the expression of Annexin-1 (Anx-1) and the transcription of its mRNA. For this purpose Anx-1 cDNA was amplified and simultaneously a T7-RNA-polymerase promotor was incorporated into the cDNA using Polymerase Chain Reaction (PCR). Then digoxigenin-11-UTP was incorporated into the transcribed cRNA with T7-RNA-polymerase. With this probe in situ hybridization was carried out in sections of the brain. The probe was visualized by an immunoassay using an anti-digoxigenin antibody conjugate. Anx-1 protein was assessed by means of immunohistochemistry using a polyclonal antibody. The various brain areas of the control animals showed an appreciable amount of Anx-1 at mRNA or protein level; on the other hand, the animals which had been pretreated with either steroid, showed a more intense Anx-1 mRNA signal than the controls in many areas. In the pretreated animals Anx-1 immunostaining was unchanged in cortex, basal ganglia, amygdala and septum, but more intense in hippocampus, hypothalamus and thalamus. In ependyma, choroid plexus, meninges, and vascular walls there was no Anx-1 mRNA transcription detectable. An opposite profile was shown by the Anx-1 immunoreactivity, the protein was present in control animals as well as the steroid-pretreated animals, suggesting that here the protein was either from systemic origin, or has diffused from adjacent structures. The results indicate that Anx-1 mRNA transcription is upregulated by either steroid, and that in the untreated animals there is a resting level of Anx-1 mRNA transcription, presumably reflecting physiological influences on Anx-1 expression.