Ghrelin and obestatin expression in oral squamous cell carcinoma: an immunohistochemical and biochemical study

The underlying molecular mechanism of carcinogenesis in oral squamous cell carcinoma (OSCC) is poorly understood and appears to be controlled on many genetic, environmental, and hormonal factors. Obestatin and ghrelin, two recently discovered hormones, are co-expressed in endocrine cells. The purpose of this investigation was to examine the immunohistochemical features of OSCCs in relation to the tissue concentration of ghrelin and obestatin. The association between OSCC and Epstein Barr Virus (EBV) status was also explored. The expression of ghrelin and obestatin was examined by immunohistochemistry and immunoassay in oral biopsy specimens: 10 benign squamous epithelial cell samples, 10 microinvasive squamous cell carcinomas, and seven well-differentiated and seven poorly differentiated OSCCs. The presence of EBV was evaluated in these samples using immunohistochemistry. The concentrations of ghrelin and obestatin in tissue homogenates were measured by RIA and ELISA, respectively. Squamous cell carcinomas and benign tissue samples were positive for anti-EBV antibody, and obestatin and ghrelin were shown to be co-expressed in all stratified squamous epithelium samples. Expression of ghrelin and obestatin was decreased or absent in OSCCs in relation to the invasiveness of the carcinoma; ghrelin and obestatin levels in cancerous tissue homogenates were lower than in benign tissue homogenates. These results indicate that the concentrations and distribution of immunoreactive obestatin and ghrelin might be helpful in distinguishing OSCC from benign tumors. Maintaining normal levels of these hormones might be required for regulation of normal cell division. However, detailed studies will be required for better understanding of the complex mechanism of carcinogenesis relating to OSCCs.     

Effect of Zinc Supplementation on Antioxidant Activity in Young Wrestlers

This study aims to examine the effect of zinc supplementation on free-radical formation and antioxidant system in individuals who are actively engaged in wrestling as a sport. The study registered a total of 40 male subjects, of whom 20 were wrestlers and 20 were sedentary individuals. The subjects were equally allocated to four groups: group 1, zinc-supplemented sportsmen group; group 2, sportsmen group without supplementation; group 3, zinc-supplemented sedentary group; group 4, sedentary group without supplementation. Blood samples were collected from all subjects twice, once at the beginning of the study and once again at the end of 8-week procedures. The blood samples collected were analyzed to determine the levels of malondialdehyde (MDA), serum glutathione (GSH), serum glutathione peroxidase (GPx) activity, serum superoxide dismutase (SOD) activity (ELISA colorimetric method) and zinc (colorimetric method). No difference was found between MDA levels of the study groups in the beginning of the study. The highest MDA value at the end of the study was obtained in group 4 (p < 0.01). MDA levels in group 2 were established to be significantly higher than those in groups 1 and 3 (p < 0.01). GSH level, GPx, and SOD activities and zinc level measured in the beginning of the study were not different between groups. Measurements performed at the end of the study showed that groups 1 and 3 (zinc-supplemented groups) had the highest GSH level, GPx, and SOD activities and zinc level (p < 0.01). These parameters were not different in the groups without supplementation (groups 2 and 4). Results obtained at the end of the study indicate that zinc supplementation prevents production of free radicals by activating the antioxidant system. In conclusion, physiologic doses of zinc supplementation to athletes may beneficially contribute to their health and performance.     

Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on human cervix epithelioid carcinoma cell line (HeLa). Nickel oxide precursors were synthesized by an nickel sulphate-excess urea reaction in boiling aqueous solution. The synthesized NiO nanoparticles (<200 nm) were investigated by X-ray diffraction analysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in 50-500 μg/mL NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy. The cytotoxicity was observed low in 50-200 μg/mL concentration for 16 h, but high in 400-500 μg/mL concentration for 2-6 h. HeLa cells' cytoplasm membrane was lysed and detached from the well surface in 400 μg/mL concentration NiO nanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in culture on the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation.     

PON1 55/192 polymorphism, oxidative stress, type, prognosis and severity of stroke

We investigated the association of PON1 55/192 polymorphisms with type, severity and prognosis of stroke and oxidative markers. Paraoxonase1 (PON1), Glutathione Reductase (GSH-Rd) and Malondialdehyde (MDA) levels were measured at day 1 and at day 5 following the onset of stroke. Genotypes were determined by polymerase chain reaction and restriction digestion. The frequencies of QQ and MM genotypes of PON1 192 and PON1 55, respectively, were significantly higher in controls than in patients. However, the allele frequencies of PON1 192 R and PON1 55 L were significantly more frequent in patients compared to controls. The frequency of combined genotype of RR/LL was significantly higher in cardioembolic group than in atherothrombotic group. PON1 activities were significantly diminished in stroke patients compared to controls. In contrast, serum MDA levels were significantly greater in patients than the values in controls. GSH-Rd activity was higher in patients with small lesion and good prognosis than those with large and poor prognosis. Low density lipoprotein (LDL) levels in patients with large lesions were higher than those with small lesions. PON1 55/192 polymorphisms influence activity of the enzyme. PON1 55/192 genotypes have been associated with MDA levels. In conclusion, PON1 genetic variations are associated with risk factors, severity, type and prognosis of stroke and oxidative stress.     

Volume-sensitive tyrosine kinases regulate liver cell volume through effects on vesicular trafficking and membrane Na+ permeability

In liver cells, the influx of Na+ mediated by nonselective cation (NSC) channels in the plasma membrane contributes importantly to regulation of cell volume. Under basal conditions, channels are closed; but both physiologic (e.g. insulin) and pathologic (e.g. oxidative stress) stimuli that are known to stimulate tyrosine kinases are associated with large increases in membrane Na+ permeability to approximately 80 pA/pF or more. Consequently, the purpose of these studies was to evaluate whether volume-sensitive tyrosine kinases mediate cell volume increases through effects on the activity or distribution of NSC channel proteins. In HTC hepatoma cells, decreases in cell volume evoked by hypertonic exposure increased total cellular tyrosine kinase activity approximately 20-fold. Moreover, hypertonic exposure (320-400 mosM) was followed after a delay by NSC channel activation and partial recovery of cell volume toward basal values (regulatory volume increase (RVI)). The tyrosine kinase inhibitors genistein and erbstatin prevented both NSC channel activation and RVI. Similarly, hypertonic exposure resulted in an increase in p60(c-src) activity, and intracellular dialysis with recombinant p60(c-src) led to activation of NSC currents in the absence of an osmolar gradient. Utilizing FM1-43 fluorescence, exposure to hypertonic media caused a rapid increase in the rate of exocytosis of approximately 40% (p < 0.01), and genistein inhibited both exocytosis and channel activation. These findings indicate that volume-sensitive increases in p60(c-src) and/or related tyrosine kinases play a key role in the regulation of membrane Na+ permeability, suggesting that increases in the NSC conductance may be mediated in part through rapid recruitment of a distinct pool of channel-containing vesicles.     

Formation of noncanonical high molecular weight caspase-3 and -6 complexes and activation of caspase-12 during serum starvation induced apoptosis in AKR-2B mouse fibroblasts

Apoptosis is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. In mammals, different complexes like the DISC complex or the apoptosome complexes have been delineated leading to the cleavage and thus activation of the executioner caspases. Although caspase-3 is the main executioner caspase in apoptosis induced by serum starvation in AKR-2B fibroblasts as demonstrated by affinity labeling with YVK(-bio)D.aomk and partial purification of cytosolic extracts by high performance ion exchange chromatography, its activation is apparently caused by a noncanonical pathway: (1) Expression of CrmA, an inhibitor of caspase-8, failed to suppress apoptosis; (2) There was no formation of high molecular weight complexes of Apaf-1 indicative for its activation. Furthermore no cleavage of caspase-9 was observed. But surprisingly, gelfiltration experiments revealed the distribution of caspase-3 and -6 into differently sized high molecular weight complexes during apoptosis. Though the apparent molecular weights of the complexes containing caspase-3 (600 kD for apoptosome and 250 kD for microapoptosome) are in accordance with recently published data, the activity profiles differ strikingly. In AKR-2B cells caspase-3 is mainly recovered as uncomplexed enzyme and in much lower levels in the apoptosomes. Remarkably, the 600 kD and 250 kD complexes containing activated caspase-3 were devoid of Apaf-1 and cytochrome c. In addition a new 450 kD complex containing activated caspase-6 was found that is clearly separated from the caspase-3 containing complexes. Furthermore, we disclose for the first time the activation of caspase-12 in response to serum starvation. Activated caspase-12 is detectable as non-complexed free enzyme in the cytosol.     

When melatonin gets on your nerves: its beneficial actions in experimental models of stroke

This article summarizes the evidence that endogenously produced and exogenously administered melatonin reduces the degree of tissue damage and limits the biobehavioral deficits associated with experimental models of ischemia/reperfusion injury in the brain (i.e., stroke). Melatonin's efficacy in curtailing neural damage under conditions of transitory interruption of the blood supply to the brain has been documented in models of both focal and global ischemia. In these studies many indices have been shown to be improved as a consequence of melatonin treatment. For example, when given at the time of ischemia or reperfusion onset, melatonin reduces neurophysiological deficits, infarct volume, the degree of neural edema, lipid peroxidation, protein carbonyls, DNA damage, neuron and glial loss, and death of the animals. Melatonin's protective actions against these adverse changes are believed to stem from its direct free radical scavenging and indirect antioxidant activities, possibly from its ability to limit free radical generation at the mitochondrial level and because of yet-undefined functions. Considering its high efficacy in overcoming much of the damage associated with ischemia/reperfusion injury, not only in the brain but in other organs as well, its use in clinical trials for the purpose of improving stroke outcome should be seriously considered.     

Effect of Intraperitoneal Selenium Administration on Liver Glycogen Levels in Rats Subjected to Acute Forced Swimming

There are a few of studies examining how selenium, which is known to reduce oxidative damage in exercise, influences glucose metabolism and exhaustion in physical activity. The present study aims to examine how selenium administration affects liver glycogen levels in rats subjected to acute swimming exercise. The study included 32 Sprague–Dawley type male rats, which were equally allocated to four groups: Group 1, general control; Group 2; selenium-supplemented control (6 mg/kg/day sodium selenite); Group 3, swimming control; Group 4, selenium-supplemented swimming (6 mg/kg/day sodium selenite). Liver tissue samples collected from the animals at the end of the study were fixed in 95% ethyl alcohol. From the tissue samples buried into paraffin, 5-μm cross-sections were obtained using a microtome, put on a microscope slide, and stained with PAS. Stained preparations were assessed using a Nikon Eclipse E400 light microscope. All images obtained with the light microscope were transferred to a PC and evaluated using Clemex PE 3.5 image analysis software. The highest liver glycogen levels were found in groups 1 and 2 (p < 0.05). The levels in group 4 were lower than those in groups 1 and 2 but higher than the levels in group 3 (p < 0.05). The lowest liver glycogen levels were obtained in group 3 (p < 0.05). Results of the study indicate that liver glycogen levels that decrease in acute swimming exercise can be restored by selenium administration. It can be argued that physiological doses of selenium administration can contribute to performance.     

Plasma serotonin levels and the platelet serotonin transporter

Serotonin (5HT) is a platelet-stored vasoconstrictor. Altered concentrations of circulating 5HT are implicated in several pathologic conditions, including hypertension. The actions of 5HT are mediated by different types of receptors and terminated by a single 5HT transporter (SERT). Therefore, SERT is a major mechanism that regulates plasma 5HT levels to prevent vasoconstriction and thereby secure a stable blood flow. In this study, the response of platelet SERT to the plasma 5HT levels was examined within two models: (i) in subjects with chronic hypertension or normotension; (ii) on platelets isolated from normotensive subjects and pretreated with 5HT at various concentrations. The platelet 5HT uptake rates were lower during hypertension due to a decrease in Vmax with a similar Km; also, the decrease in Vmax was primarily due to a decrease in the density of SERT on the platelet membrane, with no change in whole cell expression. Additionally, while the platelet 5HT content decreased 33%, the plasma 5HT content increased 33%. Furthermore, exogenous 5HT altered the 5HT uptake rates by changing the density of SERT molecules on the plasma membrane in a biphasic manner. Therefore, we hypothesize that in a hypertensive state, the elevated plasma 5HT levels induces a loss in 5HT uptake function in platelets via a decrease in the density of SERT molecules on the plasma membrane. Through the feedback effect of this proposed mechanism, plasma 5HT controls its own concentration levels by modulating the uptake properties of platelet SERT.     

At diabetes-like concentration, glucose down-regulates the placental serotonin transport system in a cell-cycle-dependent manner

Serotonin [5-hydroxytryptamine (5HT)] is a vasoconstrictor that also acts as a developmental signal early in embryogenesis. The 5HT transporter (SERT) on the membranes of the placental trophoblast cells controls 5HT levels in the maternal bloodstream to maintain stable transplacental blood flow and simultaneously provide 5HT to the embryo. The 5HT uptake rate of placental SERT is important for both the mother and the developing embryo. The impact of glucose on the placental SERT system during diabetic pregnancy is not known. The present in vitro study investigated this important issue in human placental choriocarcinoma (JAR) cells that were cultured for 24-96 h in a medium containing either 5.5 (physiologic concentration) or 25 mmol/L D-glucose (diabetic-like concentration). The 5HT uptake rates of the cultured cells were not altered at exogenous D-glucose concentrations in the range of 5.5-15 mmol/L, but were decreased significantly at a diabetic-like concentration (>or=25 mmol/L). To understand better the role of glucose on the placental 5HT system, we first characterized SERT in JAR cells at different cell-cycle phases and then determined the expression levels of SERT on the plasma membrane and in the intracellular pools of JAR cells at the late-S and G2 phases, where the uptake rates were decreased 73% under diabetic-like glucose concentrations. Finally, the importance of self-association of SERT molecules was examined. In JAR cells co-expressing Flag- and myc-tagged SERT, myc-antibody precipitated 70% of Flag-SERT, indicating that a large percentage of SERT proteins exist as oligomers in situ. Under diabetic conditions, myc-antibody no longer precipitated Flag-SERT, suggesting a disruption in the aggregation of SERT molecules. Therefore, we propose that under uncontrolled diabetic conditions, glucose down-regulates 5HT uptake rates of placental SERT by interfering with its functional expression in a cell-cycle-dependent manner.