Combining proteomics and lipid analysis to unravel Confidor stress response in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model for studying the influence of different stress factors on eukaryotic cells. In this work we used the pesticide imidacloprid, in the Confidor formulation, as the stress factor and analyzed its influence on the metabolic activity, proteome and lipid content and composition of Saccharomyces cerevisiae yeast. During the cultivation of yeast, the lowest recommended application dose of Confidor (0.025%, v/v) was added to the growth media and its influence on the mitochondria, cytosol with microsomes, and the whole yeast cells was monitored. The results show that under the stress provoked by the toxic effects of Confidor, yeast cells density significantly decreased and the percentage of metabolically disturbed cells significantly increased comparing with untreated control. Also, there was a downregulation of majority of glycolytic, gluconeogenesis, and TCA cycle enzymes (Fba1, Adh1, Hxk2, Tal1, Tdh1,Tdh3, Eno1) thus providing enough acetyl‐CoA for the lipid restructuring and accumulation mechanism since we have found the changes in the cell and mitochondrial lipid content and FA composition. This data suggest that lipids could be the molecules that orchestrate the answer of the cells in the stress response to the Confidor treatment.     

Central nervous system-infiltrated immune cells induce calcium increase in astrocytes via astroglial purinergic signaling

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naïve astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell–astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca²⁺ increase in astrocytes by promoting glial release of ATP. ATP propagated Ca²⁺ elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca²⁺ increase is specifically triggered by the autoreactive CD4⁺ T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca²⁺ signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell–astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease.     

Cell cycle analysis of the CD133+ and CD133- cells isolated from umbilical cord blood

BACKGROUND: Umbilical cord blood cells (stem/progenitor cells) exhibit high proliferative capacities leading to a large expansion of cells in appropriate cell culture conditions. The aim of this study was to evaluate by flow cytometry the cycling status of CD133+ and CD133- cells depending on various culture conditions, such as sera, stem cell factor (SCF), interleukin 3 (IL-3) and interleukin 6 (IL-6). METHODS: An immunomagnetic system was used for cell separation. CD133+ and CD133- cells were seeded in Iscove's Modified Dulbecco's Medium (IMDM) with different serum concentrations and were stimulated with SCF (100 ng/ml), IL-3 (50 ng/ml) and IL-6 (50 ng/ml). RESULTS: Our experiments demonstrated that immediately after separation, 96.75+/-0.58% of CD133+ cells and 97.04+/-1.76% of CD133- cells were in G0/G1-phase, while 2.02+/-0.38% and 0.88+/-0.52% were in the S-phase, respectively. Our data documented that CD133+ cells are more active than CD133- cells after the first week of cultivation (p<0.01). Statistically significant difference was found for CD133+ cells vs. CD133- cells after second week of cultivation in G0/G1- and S-phases under all tested conditions. A combination of 12.5% FCS+12.5% HS yielded the highest cell expansion for CD133+ cells; this was concomitant with highest percentage of S-phase and G2M-phase. Our data show that the medium with 25% HS was the best for cell expansion and cycling of the CD133- cells for the first week, followed by the 12.5% FCS+12.5% HS. After 2 weeks of cultivation, obviously 12.5% HS and 12.5% FCS+12.5% HS exhibited similar S-phase amounts in CD133- cells. A decrease of HS concentrations seemed to stimulate CD133- cells' S-phase after the second week. CONCLUSIONS: Our data indicate that the source and the concentration of the serum used for cultivation have an impact on both cell populations: CD133+ cells are most comfortable with a combination of FCS and HS; CD133- cells prefer media-containing HS. Cell cycle status may be an important factor for defining cultivation strategies for stem cell expansion.     

Tamoxifen stimulates calcitonin-producing thyroid C-cells and prevents trabecular bone loss in a rat model of androgen deficiency

Thyroid C-cells produce calcitonin (CT), a hypocalcemic hormone, that acts as an inhibitor of bone resorption. In this study, we investigated the effects of tamoxifen (TAM) as a selective estrogen receptor modulator on thyroid C-cells, trabecular bone and biochemical markers of bone metabolism in an animal model of androgen deficiency, represented by middle-aged orchidectomized (Orx) rats. Fifteen-month-old male Wistar rats were divided into: Orx and sham-operated (SO) groups. Rats from one Orx group were injected subcutaneously with TAM citrate (Orx + TAM; 0.3 mg kg⁻¹ b.w.), while the rats from SO and a second Orx group received vehicle alone, once a day for 3 weeks. The peroxidase–antiperoxidase method was applied for localization of CT in C-cells. Thyroid C-cells were morphometrically and ultrastructurally analyzed. An ImageJ image-processing program was used to measure bone histomorphometric parameters. Blood serum samples were analyzed for CT, osteocalcin (OC), calcium (Ca²⁺) and phosphorus (P). Urinary Ca²⁺ concentrations were measured. TAM treatment significantly increased thyroid C-cell volume (V_c) and serum CT when compared with vehicle-treated Orx rats. Analysis of trabecular microarchitecture of the tibia showed that administration of TAM significantly increased cancellous bone area, trabecular thickness and trabecular number, whereas trabecular separation was significantly decreased compared with vehicle-treated Orx rats. Serum OC and urinary Ca²⁺ concentrations were significantly lower in comparison with the control Orx group. These results indicate that in our rat model of androgen deficiency, TAM stimulated calcitonin-producing thyroid C-cells and increased trabecular bone mass.     

Exposure to dexamethasone reduces pituitary volume and gonadotropic cell number in rat fetuses

Overexposure to glucocorticoids during the fetal period induces changes in developmental processes in various fetal tissues. The aim of this study was to investigate the effects of the synthetic glucocorticoid, dexamethasone (Dx), on pituitary volume and gonadotropic cells during a critical period of pituitary development. The effects of Dx on stereological parameters of the pituitary gland and FSH and LH cells were investigated in 19 and 21-day old fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg Dx/kg b.w. subcutaneously, followed by 0.5 mg Dx/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline. FSH and LH cells were stained immunohistochemically by the peroxidase–antiperoxidase method (PAP). In 19-day old fetuses, exposure to Dx caused a significant decrease of pituitary volume, estimated by Cavalieri's principle. Also, the total number of FSH and LH cells per pituitary, determined by physical fractionator counting technique, was significantly reduced. These changes persisted until fetal day 21. Volume densities and numerical densities of FSH and LH cells after exposure to Dx in 19 and 21-day old fetuses remained unaffected. Our results suggest that altered stereological parameters in pituitary gland after exposure to dexamethasone in fetal period could be long-lasting.