Maja Milošević

17β-estradiol modulates mitochondrial Ca2+ flux in rat caudate nucleus and brain stem

The aim of this study was to examine the rapid non-genomic effect of 17β-estradiol (E2) on Ca(2+) transport in mitochondria isolated from the nerve terminals (synaptosomes) of caudate nuclei (NC) and brain stems (BS) of ovariectomised female rats. In physiological conditions no effect of E2 on Ca(2+) influx into synaptosomal mitochondria through ruthenium red (RR)-sensitive uniporter was observed. However, in the presence of uncoupling agent carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) (1μmol/l), pre-treatment with 0.5nmol/l E2 protected mitochondrial membrane potential and consequently increased Ca(2+) influx (2.3-fold in NC and 3.1-fold in BS). At the same time, 0.5nmol/l E2 by increasing the affinity of mitochondrial Na(+)/Ca(2+) exchanger for Na(+) inhibited mitochondrial Ca(2+) efflux in NC and BS by about 40%. Also, the specific binding of physiological E2 concentrations (0.1-10nmol/l) to isolated synaptosomal mitochondria was detected. Using membrane impermeable E2 bound to bovine serum albumin and selective inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, we obtained that E2s action on mitochondrial Ca(2+) efflux at least partially is due to the direct effects on the mitochondrial membrane and/or Na(+)/Ca(2+) exchanger located in inner mitochondrial membrane. Our results implicate E2 as a modulator of Ca(2+) concentration in mitochondrial matrix, and ultimately in the cytosol. Given the vital role of Ca(2+) in regulation of total nerve cells activity, especially energy metabolism, neurotransmission and directing the cells toward survival or cell death, the effects on mitochondrial Ca(2+) transport could be one of the important modes of E2 neuromodulatory action independent of the genome.

Diffuse reflectance spectra of methylene blue adsorbed on different types of clay samples

Abstract Diffuse reflectance (DR) spectra in the visible (VIS) and near-infrared (NIR) region of methylene blue (MB) adsorbed on clay samples of different mineral composition and varying MB/clay ratios were analyzed. This type of investigation is not affected by unadsorbed MB molecules and can be used in clay-mineral determination. When the MB dye/clay loading was increased, enhancement of bands in the DR-VIS and DR-NIR was observed. The distribution of various MB aggregates was interpreted according to mineral composition and MB loading. The NIR spectrum of H2O overtone provides insights about the intercalated MB aggregation, consistent with VIS spectra. Critical MB concentration for formation of dimeric structure was between 0.7 and 1.75 mmol/100 g followed with suppression of monomers. Different orientations of vectors of polarized light allowed calculations of the possible orientation and tilting angle of MB molecules on the surface of the clay particles in accordance with XRD data.

Inhibition of mitochondrial Na+-dependent Ca2+ efflux by 17β-estradiol in the rat hippocampus

Our results, as well as those of others, have indicated that 17β-estradiol (E2) exerts its nongenomic effects in neuronal cells by affecting plasma membrane Ca(2+) flux. In neuronal cells mitochondria possess Ca(2+) buffering properties as they both sequester and release Ca(2+). The goal of this study was to examine the rapid non-genomic effect of E2 on mitochondrial Ca(2+) transport in hippocampal synaptosomes from ovariectomised rats. In addition, we aimed to determine if, and to what extent, E2 receptors participated in mitochondrial Ca(2+) transport modulation by E2 in vitro. E2-specific binding and Ca(2+) transport was monitored. At physiological E2 concentrations (0.1-1.5 nmol/L), specific E2 binding to mitochondria isolated from hippocampal synaptosomes was detected with a B(max.) and K(m) of 37.6±2.6 fmol/mg protein and 0.69±0.14 nmol/L of free E2, respectively. The main mitochondrial Ca(2+) influx mechanism is the Ruthenium Red-sensitive uniporter driven by mitochondrial membrane potential. Despite no effect of E2 on Ca(2+) influx, a physiological E2 concentration (0.5 nmol/L) protected mitochondrial membrane potential and consequently Ca(2+) influx from the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (1 μmol/L). In neuronal cells the predominant mitochondrial Ca(2+) efflux mechanism is the Na(+)/Ca(2+) exchanger. E2 caused Ca(2+) efflux inhibition (by 46%) coupled with increased affinity of the Na(+)/Ca(2+) exchanger for Na(+). Using E2 receptor (ERα and ERβ) antagonists and agonists, we confirmed ERβs involvement in E2-induced mitochondrial membrane potential protection as well as Ca(2+) efflux inhibition. In summary, our results indicate that the non-genomic neuromodulatory role of E2 in rat hippocampus is achieved by affecting mitochondrial Ca(2+) transport via, in part, mitochondrial ERβ.

Effect of acute heat stress on rat adrenal medulla — a morphological and ultrastructural study

Isolated rat adrenal medulla was analyzed by light and electron microscope after an acute (60 min) exposure to high ambient temperature (38°C). Under these conditions there was a significant rise in plasma adrenaline and noradrenaline. Stereological investigation by light microscopy showed a significant decrease in volume density of cells and an increase in the interstitium. At the ultrastructural level, the profile area of cells, nuclei and cytoplasm of adrenaline cells were significantly decreased. After the heat stress numbers of resting granules in adre naline and noradrenaline cells were significantly reduced, while the numbers of altered granules and empty containers in both types of adrenomedullar cells were significantly increased.

Effects of acute gamma-irradiation on extracellular adenine nucleotide hydrolysis in developing rat brain

Cell membrane is highly sensitive to irradiation which, acting directly or indirectly, may disturb functions of constitutive proteins including membrane enzymes. Plasma membrane surface-located enzyme chain of ecto-nucleotide triphospho diphosphohydrolases (NTPDases) and 5′-nucleotidase are involved in termination of cell purinergic signalization by hydrolyzing extracellular, excitatory adenosine triphosphate (ATP), as well as nucleotide di-, and mono-phosphate (ADP and AMP) to neuroprotective adenosine. Extracellular ATP, ADP, and AMP hydrolyzes were examined in purified synaptic plasma membranes after whole-body acute irradiation. All measurements were done 24 h after irradiation of developing (15-, 30-day-old) and adult (90-day-old) rats with low (50 cGy) and high (2 Gy) dose of gamma-rays. Both, high and low doses inhibited nucleotide hydrolyses in 15-day-old rats; in 30-day-old rats low dose of radiation inhibited ADP and AMP hydrolyses while high dose inhibited only ATP hydrolyse. In adult rats high dose induced no effects, while low dose stimulated nucleotides hydrolyses. According to obtained results it was concluded that ecto-nucleotidases of young rats are more sensitive to irradiation, since even low dose induces inhibition of ecto-nucleotidases activities. Ionizing radiation, by decreasing brain nucleotide hydrolyses in developing rats, induces accumulation of ATP and decreases production of adenosine in synaptic cleft which could be neurocytotoxic. On the contrary, in adult rats low dose of radiation stimulates NTPDase and 5′-nucleotidase activity and protective adenosine production which indicates protective and adaptive mechanisms developed in adult brain neuronal cells.

Plasma phospholipid changes are associated with response to chemotherapy in non-Hodgkin lymphoma patients

Limited studies have been performed to associate abnormal phospholipid (PL) profile and disease activity in hematological malignancies, including non-Hodgkin lymphoma (NHL). The aim of his study was to evaluate the levels of plasma PL fractions in NHL patients, in response to chemotherapy. Forty non-treated patients with NHL and 25 healthy individuals were recruited. Blood samples from patients were taken before chemotherapy, after 3 cycles and after the end of the treatment, and PL fractions were resolved by one-dimensional thin-layer chromatography. To assess potential relationship between plasma PL profile and response to therapy, patients were divided according to clinical outcome in 3 groups: complete remission (CR), stable disease (SD) and progression (PG). Despite significant differences between NHL patients and healthy controls, no differences were found at baseline among patients divided according to clinical outcome. During and after chemotherapy important alterations in PL profile were observed. Levels of total PLs and all PL fractions decreased in patients with PG while in patients who responded to therapy (CR, SD) PLs significantly increased. Results of our study suggest that changes of total PLs and PL fractions during the therapy are associated with the effects of therapy and clinical outcome in patients with NHL.

Kinetic characterization of ecto-nucleoside triphosphate diphosphohydrolases in brain nerve terminals during rat postnatal development

A family of enzymes named ecto-nucleoside triphosphate diphosphohydrolase (NTPDases) catalyzes the termination of ATP and ADP actions. Three different NTPDases (NTPDase 1–3), differing in their preference for a substrate, have been localized in the brain of adult mammals. The goal of our study was to clarify ATP and ADP hydrolyzing activities and kinetic parameters of NTPDases in synaptic plasma membranes (SPM) isolated from 15-, 30-, 60- and 90-days-old female rat brains. ATP and ADP hydrolysis were maximal in the presence of Mg2+ and showed insensitivity to ion-transporting ATPase inhibitors. The pronounced increase in both, ATP and ADP hydrolysis, were found in the SPM isolated from rats in the first month of life, stayed at the same level in the second month, and then decreased in adulthood. Kinetic analysis are also developmental-dependent, and together with the rate of ATP:ADP hydrolysis, point that all three NTPDases are present in SPM isolated from different developmental stages, with different, developmental-dependent proportion of activities. The lowest velocity and the highest affinity were observed for ATP hydrolyses, while the highest velocity and lowest affinity were detected for ADP hydrolyses in SPM isolated from 15-day old rats. Since specific ATP and ADP hydrolysis were lowest in this stage, we concluded that velocity is crucial for ATPase-, while affinity is for ADPase-part of NTPDases. Increased NTPDases activities, changes in their hydrolysis velocity and substrates affinities during rat postnatal development indicate involvement of adenine nucleotides in processes implicated to neuronal maturation and augmented neuroprotection.

Cholecystokinin-producing (I) cells of intestinal mucosa in dexamethasone-treated rats.

The aim of this study was to investigate the morphological, immunohistochemical and ultrastructural changes of cholecystokinin-producing (I) cells of gastrointestinal mucosa in dexamethasone-treated rats (D). After 12-daily intraperitoneal administration of 2mg/kg dexamethasone, rats developed diabetes similar to human diabetes mellitus type 2. The mean diameter of the duodenum was significantly decreased due to significant reduction of volume fraction and profile area of lamina propria. There was a decrease in volume fraction and number of cholecystokinin (CCK)-producing cells per mm(2) of mucosa, as well as their numerical density, but without statistical significance. Also, dexamethasone induced appearance of hyperactive duodenal I-cells with small number of granules and dilated endoplasmic reticulum. In conclusion, the present study showed that morphological changes in duodenum cholecystokinin-producing (I) cells occurred in diabetic rats, in a manner which, suggests compensatory effort of CCK cells in diabetic condition.

Effect of EDTA on copper-induced inhibition of rat myometrial ecto-ATPase activity

AbstactThe aim of this study was to examine in vitro chelator’s ability to prevent copper-induced inhibition of rat myometrial ecto-ATPase activity. The effects of increasing CuSO4 concentrations, in the absence and presence of 1 mmol/l EDTA, showed sigmoidal and complete inhibition relative to the control enzyme activity. IC50 values, 1.15 × 10−4 and 1.71 × 10−3 mol/l in the absence and presence of EDTA, respectively, were determined by Hill analysis from experimental curves. According to the results presented in this work, 1 mmol/l EDTA increased by one order of magnitude CuSO4 concentration for half-maximal inhibition (IC50), by decreasing Cu2+ concentrations, available to form inactive CuATP2− complex.

Characterization of stem-like cancer cells in basal cell carcinoma and its surgical margins

Understanding the pathogenesis of basal cell carcinoma (BCC) and identifying the cells responsible for propagation and recurrence are crucial for the development of new treatment strategies. The aim of this study was to characterize the cells isolated from BCC and its margin.