Blood pressure modulation following activation of mast cells by cationic cell penetrating peptides

Short cell penetrating peptides (CPP) are widely used in vitro to transduce agents into cells. But their systemic effect has not been yet studied in detail. We studied the systemic effect of the cell penetrating peptides, penetratin, transportan and pro-rich, on rat hemodynamic functions. Intra-arterial monitoring of blood pressure showed that injection of the positively charged penetratin and transportan in a wide range of concentrations (2.5-320 μg/kg) caused highly significant transient decrease in the systolic and diastolic blood pressure in a dose dependent manner (p<0.01). Pretreatment with histamine receptors blockers or with cromolyn, a mast cell stabilizing agent, significantly attenuated this effect. Furthermore, in vitro incubation of these both peptides with mast cells line, LAD2, caused a massive mast cell degranulation. In vitro studies showed that these CPP in a wide range of concentrations were not cytotoxic without any effect on the survival of LAD2 mast cell line. In contrast, the less positively charged and proline-rich CPP, pro-rich, had no systemic effects with no effect on mast cell degranulation. Our results indicate that intravenously administrated positively charged CPP may have deleterious consequences due to their induced BP drop, mediated by mast cell activation. Therefore, the major effect of mast cell activation on BP should be considered in developing possible future drug therapies based on the injection of membrane-permeable and positively charged CPP. Nevertheless, lower levels of such CPP may be considered as a treatment of systemic high BP through moderate systemic mast cell activation.     

Expression of Stem Cell Markers in the Human Fetal Kidney

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34^th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms'' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms'' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAM^neg and EpCAM^dim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24⁺CD133⁺ cells comprise >50% of HFK cells and predominantly co-express EpCAM^bright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM⁺EpCAM^- and to a lesser extent in NCAM⁺EpCAM⁺ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM⁺EpCAM⁺FZD7⁺), MM stem cells (NCAM⁺EpCAM^-FZD7⁺) or both (NCAM⁺FZD7⁺). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.     

Cardioprotective and antioxidant effects of apomorphine

Apomorphine is a potent antioxidant that infiltrates through biological membranes. We studied the effect of apomorphine (2 microM) on myocardial ischemic-reperfusion injury in the isolated rat heart. Since iron and copper ions (mediators in formation of oxygen-derived free radicals) are released during myocardial reperfusion, apomorphine interaction with iron and copper and its ability to prevent copper-induced ascorbate oxidation were studied. Apomorphine perfused before ischemia or at the commencement of reperfusion demonstrated enhanced restoration of hemodynamic function (i.e. recovery of the work index (LVDP x HR) was 69.2 +/- 4.0% with apomorphine pre-ischemic regimen vs. 43.4 +/- 9.01% in control hearts, p < 0.01, and 76.3 +/- 8.0% with apomorphine reperfusion regimen vs. 30.4 +/- 11.1% in controls, p < 0.001). This was accompanied by decreased release of proteins in the effluent and improved coronary flow recovery in hearts treated with apomorphine after the ischemia. Apomorphine forms stable complexes with copper and with iron, and inhibits the copper-induced ascorbate oxidation. It is suggested that these iron and copper chelating properties and the redox-inactive chelates formed by transition metals and apomorphine play an essential role in post-ischemic cardioprotection.     

Biochemical and Molecular Responses to Loss of Renal Mass: Membranes and Protein Synthesis: Further Studies on a Renotropic System in Rats

Based upon many investigations, the existence of short-lived, specific, circulating substances which incite and/or regulate compensatory renal growth has been proposed. In our studies, we find that sera and plasma from unilaterally nephrectomized rats compared to sera and plasma from sham-operated rats stimulate the incorporation of ³H-thymidine monophosphate, ³H-thymidine and ¹⁴C-uridine into the DNA of incubating rat kidney fragments. While extracts from growing rat kidneys are not excitatory, they produce a relative enhancement to incorporation of isotope into DNA when combined with sera from uninephrectomized rats—more than the sera do alone. The above is found also for the incorporation of ¹⁴C-uridine into RNA of incubating rat kidney fragments. Sera from uninephrectomized rats fail to stimulate DNA synthesis in liver slices from rats but do so in the presence of extracts from growing kidneys. Renotropic factors in sera and extracts do not appear to work by diluting the isotopes, by enhancing transport, or by effecting overall metabolism of the renal cells. The above described serum and liver factors may play a role in compensatory renal growth.     

Cardioprotective and Antioxidant Effects of Apomorphine

Apomorphine is a potent antioxidant that infiltrates through biological membranes. We studied the effect of apomorphine (2?μM) on myocardial ischemic-reperfusion injury in the isolated rat heart. Since iron and copper ions (mediators in formation of oxygen-derived free radicals) are released during myocardial reperfusion, apomorphine interaction with iron and copper and its ability to prevent copper-induced ascorbate oxidation were studied. Apomorphine perfused before ischemia or at the commencement of reperfusion demonstrated enhanced restoration of hemodynamic function (i.e. recovery of the work index (LVDP?×?HR) was 69.2±4.0% with apomorphine pre-ischemic regimen vs. 43.4±9.01% in control hearts, p<0.01, and 76.3±8.0% with apomorphine reperfusion regimen vs. 30.4±11.1% in controls, p<0.001). This was accompanied by decreased release of proteins in the effluent and improved coronary flow recovery in hearts treated with apomorphine after the ischemia. Apomorphine forms stable complexes with copper and with iron, and inhibits the copper-induced ascorbate oxidation. It is suggested that these iron and copper chelating properties and the redox-inactive chelates formed by transition metals and apomorphine play an essential role in post-ischemic cardioprotection.     

Increased frequency of oxidant-mediated DNA strand breaks in mononuclear leucocytes exposed to activated neutrophils from cigarette smokers

Following activation with the synthetic chemotactic tripeptide FMLP, potentiated by cytochalasin B (CB), blood neutrophils from cigarette smokers generated greater amounts of both extracellular and intracellular reactive oxidants than cells from non-smoking control subjects. FMLP/CB-activated neutrophils from cigarette smokers also inflicted increased oxidant-mediated damage to the DNA of cocultured mononuclear leucocytes, which was prevented by the inclusion of superoxide dismutase and catalase individually and in combination. These observations demonstrate that cigarette smoking primes phagocytes to generate increased amounts of potentially carcinogenic reactive oxidants.     

Nicorandil decreases postischemic actin oxidation

This study examined the hypothesis that preconditioning can decrease postischemic oxidative protein damage. Isolated rat hearts were subjected to 25 min of normothermic global ischemia followed by 45 min of reperfusion. These were compared with hearts pretreated with 20 microM nicorandil or preconditioned with two cycles of ischemia. Changes in the high energy phosphates, ATP and phosphocreatine, were followed using (31)P-NMR spectroscopy. Protein carbonyls were assessed using an immunoblot technique. Postischemic hemodynamic function and high energy phosphates recovered to significantly (p <.05) higher levels in nicorandil-treated and ischemic preconditioned hearts as compared to controls. Postischemic protein carbonyl formation was highest in control reperfused hearts but reduced to intermediate between control and preischemic hearts by ischemic preconditioning and virtually prevented by nicorandil pretreatment, with a prominent band at 43 kDa significantly affected (p <.05). Based on immunoshift and immunoprecipitation studies, this band was identified as a mixture of actin isoforms. These studies support the conclusion that nicorandil diminishes protein oxidative damage in general, and specifically actin oxidation, which in the presence of improved supply of high energy phosphates, leads to enhanced postischemic contractile function.