Stimulation of Plastidogenesis Induced by 5-Azacytidine in Euglena gracilis Klebs

When etiolated Euglena gracilis was treated with 10 mM 5-azacytidine (5-azaC), an inhibitor of DNA methylation, stimulation of plastidogenesis in both dark and light conditions was observed. The phenomenon occurred in 10–15% of the cells possibly due to the asynchronicity of the cultures. The main features of this sub-population, as evaluated by electron and fluorescence microscopy, were the following: 1. the presence in darkness of differentiating proplastids that were red fluorescent under UV, positive to TCNBT cytochemical reaction (specific for PSII) and negative to DAB (specific for PSI); 2. the acceleration of proplastid differentiation during the first 20–30 h of illumination; 3. the occurrence in both culture conditions of concentric lamellar bodies (LB_S). These structures were considered to be proplastids blocked in the first step of evolution, since they emitted a red fluorescence, were contained within compartments limited by a triple-layered envelope, were reactive to TCNBT in darkness and to both TCNBT and DAB in light conditions. Even if the action mechanism of 5-azaC on plastidogenesis in Euglena remains to be defined, the induced stimulatory effect on plastid differentiation pointed to a relationship between DNA methylation and plastid development. Furthermore, the presence of LB_S opens the possibility of studying early aspects of plastid development in Euglena.     

Do benthic and planktonic diatoms produce equivalent effects in crustaceans?

Hippolyte inermis Leach 1814 is a benthic shrimp characterized by a peculiar mechanism of sex reversal influenced by diatom foods. In fact, the appearance of primary females in spring is due to an apoptotic early disruption of the androgenic gland and of the male gonad, triggered by still unknown compounds present in diatoms of the genus Cocconeis. The influence of diatoms on the reproductive ecology and life cycle of planktonic crustaceans has been demonstrated previously: some planktonic diatoms produce aldehydes inducing apoptosis in the embryos and in the larvae of marine copepods, reducing their viability. Both benthic and planktonic diatoms therefore produce compounds having an apoptotic effect on some tissues of target crustaceans, although the ecological significance of the two processes is different: deleterious for copepod populations, regulative for shrimps associated with Posidonia oceanica. In the present article we experimentally administered specific planktonic diatoms, their fractions and compounds known to induce apoptosis in planktonic copepods, to H. inermis postlarvae, to check whether the apoptotic effect is due to an identical family of diatom compounds, and to establish whether the processes observed in the plankton and in the benthos, respectively, are analogous or homologous, from an ecological point of view. Our results indicated that diatom compounds acting in the two systems are different, since both planktonic diatoms and their aldehydes had negligible effects on the sex ratios of cultured shrimps.     

Nitric oxide-mediated arteriolar dilation after endothelial deformation

Previously, we frequently observed dilation of arterioles after agonist-induced constrictions. We hypothesized that deformation of the endothelium during decreases in diameter of isolated arterioles elicits the release of nitric oxide (NO). In isolated arterioles of rat mesentery, phenylephrine (PE, 10(-7) M)-, U-46619 (10(-7) M)-, and KCl (50 mM)-induced constrictions were followed by potent dilations. Inhibition of NO synthase with N(omega)-nitro-L-arginine (L-NNA, 2 x 10(-4) M) or removal of the endothelium significantly enhanced constriction and reduced the postconstriction dilation. In the presence of 80 mmHg of intraluminal pressure, an increase in extraluminal pressure (P(e)) to 75 mmHg for 20 s and 1 and 2 min decreased vessel diameter. After release of P(e), arterioles dilated as a function of the duration of diameter reduction by P(e). Removal of the endothelium or administration of L-NNA significantly diminished the post-P(e) dilations. In cultured mesenteric arteriolar endothelial cells (EC), PE, U-46619, or KCl did not increase, whereas ACh did increase, the production of NO, as measured by a fluorometric assay for nitrite. Furthermore, when EC, cultured on a stretched silicone membrane, were subjected to deformation by shortening the membrane to 50% of its original length, NO release increased significantly. Based on all of the above, we propose that deformation of EC per se elicits release of NO, a mechanism that modulates arteriolar constriction.     

Ion cyclotron resonance as a tool in regenerative medicine

The identification of suitable stem cell cultures and differentiating conditions that are free of xenogenic growth supplements is an important step in finding the clinical applicability of cell therapy in two important fields of human medicine: heart failure and bone remodeling, growth and repair. We recently demonstrated the possibility of obtaining cardiac stem cells (CSCs) from human endomyocardial biopsy specimens. CSCs self-assemble into multi-cellular clusters known as cardiospheres (CSps) that engraft and partially regenerate infarcted myocardium. CSps and cardiosphere-derived-cells (CDCs) were exposed for five days in an incubator regulated for temperature, humidity, and CO(2) inside a solenoid system. This system was placed in a magnetically shielded room. The cells were exposed simultaneously to a static magnetic field (MF) and a parallel low-alternating frequency MF, close to the cyclotron frequency corresponding to the charge/mass ratio of the Ca(++) ion. In this exposure condition, CSps and CDCs modulate their differentiation turning on cardiogenesis and turning off vasculogenesis. Cardiac markers such as troponin I (TnI) and myosin heavy chain (MHC) were up-regulated. Conversely, angiogenic markers such as vascular endothelial growth factor (VEGF) and kinase domain receptor (KDR) were down-regulated as evidenced by immunocytochemistry. Exposure to the 7 Hz calcium ion cyclotron resonance (ICR) frequency can modulate the cardiogenic vs. angiogenic differentiation process of ex vivo expanded CSCs. This may pave the way for novel approaches in tissue engineering and cell therapy. With regard to bone remodeling, it has been suggested that bone marrow-derived mesenchymal stem cells (MSC) may be considered as a potential therapeutic tool. Using the Ca(++)-dependent specific differentiation potential of the ELF-MF 7 Hz ICR, we show here that exposure of human MSC to these same MF conditions enhanced the expression of osteoblast differentiation markers such as alkaline phosphatase, osteocalcin, and osteopontin, as analyzed by real-time quantitative PCR, without affecting cell proliferation. As expected, while the differentiation marker factors were up regulated, the ICR electromagnetic field down regulated osteoprotegerin gene expression, a critical regulator of postnatal skeletal development and homeostasis in humans as well as mice.