Investigation of modified platelet-rich plasma (mPRP) in promoting the proliferation and differentiation of dental pulp stem cells from deciduous teeth

Stem cells from human exfoliated deciduous teeth (SHEDs) have great potential to treat various dental-related diseases in regenerative medicine. They are usually maintained with 10% fetal bovine serum (FBS) in vitro. Modified platelet-rich plasma (mPRP) would be a safe alternative to 10% FBS during SHEDs culture. Therefore, our study aimed to compare the proliferation and differentiation of SHEDs cultured in mPRP and FBS medium to explore an optimal concentration of mPRP for SHEDs maintenance. Platelets were harvested by automatic blood cell analyzer and activated by repeated liquid nitrogen freezing and thawing. The platelet-related cytokines were examined and analyzed by ELISA. SHEDs were extracted and cultured with different concentrations of mPRP or 10% FBS medium. Alkaline phosphatase (ALP) activity was measured. Mineralization factors, RUNX2 and OCN, were measured by real-time PCR. SHEDs were characterized with mesenchymal stem cells (MSCs) markers including vimentin, CD44, and CD105. mPRP at different concentrations (2, 5, 10, and 20%) enhanced the growth of SHEDs. Moreover, mPRP significantly stimulated ALP activity and promoted expression of RUNX2 and OCN compared with 10% FBS. mPRP could efficiently facilitate proliferation and differentiation of SHEDs, and 2% mPRP would be an optimal substitute for 10% FBS during SHEDs expansion and differentiation in clinical scale manufacturing.

Effect of gallic acid on alkaline phosphatase and peptidase activities in rat intestine.

Gallic acid is a normal constituent of many edible foods, thus directly interacts with epithelial tissue in intestine. In the present study, the effect of gallic acid on intestinal alkaline phosphatase (IAP) and peptidase activities in rat intestine was evaluated. Gallic acid (0.27-0.5 mM) inhibited activities of leucine aminopeptidase (LAP) and -glutamyl transpeptidase (-GTP) by over 90%, compared to controls in rat intestine. In contrast, 0.1-0.6 mM gallic acid either had no effect or stimulated the activity of IAP in rat intestine. The observed inhibition of peptidases by gallic acid was reversible in nature. Kinetic analysis revealed no change in Vmax of LAP (0.42-0.44 units/mg protein) and -GTP (0.22-0.24 units/mg protein), while the values of apparent Km were increased 6-7 fold, exhibiting competitive-type of enzyme inhibition by gallic acid. The values of Ki for LAP and -GTP were 0.037 mM and 0.017 mM, respectively. These observations indicate that gallic acid is a potent inhibitor of brush border peptidases, and thus may interfere in the digestion and absorption of proteins in the intestine.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84 percent, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11 percent, respectively). â-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36 percent, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18 percent) and inhibited (13 percent) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

In vitro modulation of alkaline phosphatase activity of Saccharomyces cerevisiae grown in low or high phosphate medium

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186 percent, L-leucine _ 155 percent and L-phenylalanine - 168 percent) and with 1 mM levamisole (122 percent; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20 percent, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166 percent) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90 percent, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

Inhibitory effects of some substrate level analogues on salp-activity in B-thalassaemia

Higher levels of SALP-activity was recorded in serum of patients with a-thalassaemia in comparison with normal control Subjects at specific PH and temp. Both kinetic constant, Km and Vmax has been determined. Inhibitory effects of three substrate analogues, L-Phe, Adenosine, and inorganic phosphate were investigated. Types and degree of inhibition were determined together with values of Ki [inhibitory constant] using Dixons plot

Hepatoprotective effect of Himoliv, a polyherbal formulation in rats.

The effect of Himoliv (HV) was evaluated in carbon tetrachloride or paracetamol induced hepatotoxicity in rats. Liver necrosis was produced by administering single dose of either carbon tetrachloride (CCl4, 1 ml/kg, 50% v/v with olive oil, s.c.) or paracetamol (PC, 1 g/kg, p.o.). The liver damage was evidenced by elevated levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and serum alkaline phosphatase (ALP) and hepatic thiobarbituric acid reacting substances (TBARS) and superoxide dismutase (SOD). HV pretreatment (0.5 and 1.0 ml/kg, p.o.) significantly (P < 0.001) reduced CCl4 or PC-induced elevations of the levels of SGOT, SGPT, ALP and TBARS, while the reduced concentration of SOD due to CCl4 or PC was reversed. Silymarin (25 mg/ kg, p.o.), a known hepatoprotective drug showed similar results.

Modulatory effect of Mentha piperita (Linn.) on serum phosphatases activity in Swiss albino mice against gamma irradiation.

Mentha extract (ME; 1 g/kg body wt) given orally for three consecutive days prior to whole body irradiation (8 Gy) showed modulation of activity of serum phosphatases in albino mice. Values of acid phosphatase activities were significantly higher in untreated irradiated group throughout the experiment. Irradiated animals pretreated with ME showed significant decline in acid phosphatase activity as compared to untreated irradiated animals at all autopsy intervals and attained normalcy at day 5. A marked decrease in serum alkaline phosphatase activity was recorded in both irradiated groups. However, in ME pretreated irradiated group, values of alkaline phosphatase activity remained significantly higher than untreated irradiated animals at all intervals and attained normalcy from day 5 onwards.

A simple in vitro method to detect singlet oxygen and to compare photodynamic activity using alkaline phosphatase.

A simple, sensitive and reliable in vitro method based on photodynamic inactivation of alkaline phosphatase to detect singlet oxygen and for evaluating relative photosensitizing efficiencies of photosensitizers such as hematoporphyrin (Hp) and phthalocyanines has been developed and compared with photobleaching of p-nitroso dimethyl aniline (RNO) and photooxidation of L-tryptophan. Inactivation of alkaline phosphatase is dependent both on light fluence and sensitizer concentration. Scavengers like mannitol and azide anion indicated the involvement of singlet oxygen in the deactivation of alkaline phosphatase, since azide anion provided concentration dependent protection whereas mannitol had no effect and that compared to ordinary water, photoinactivation of alkaline phosphatase was three times higher in 65% D2O. Alkaline phosphatase appears to be resistant to free radical attack (particularly to OH radicals) since hydrogen peroxide alone or in presence of ferrous ions did not reduce the enzyme activity and mannitol or azide anion gave no significant protection when alkaline phosphatase was irradiated with Co-60 gamma rays up to 2 K Gy. With the present method using red light, the chloroaluminium phthalocyanine sulphonates prepared by sulphonation showed higher and the corresponding condensation product lower photodynamic activity; Hp being intermediate and Mn- and Gd-phthalocyanines had no photodynamic activity.

Toxic effects of garlic extract and garlic oil in rats.

Significant rise in urea and D-aspartate aminotransferase and inhibition of alkaline phosphatase in serum were observed in rats fed garlic extract (2 ml/100 g body wt, intragastrically) for 10 days. The liver showed histological changes. Garlic oil feeding (10 mg/100 g body wt, intragastrically) after 24 hr fasting was found lethal. The cause of death appears to be acute pulmonary oedema. On histological examination, all the organs of the dead rats revealed severe congestion. However, similar feeding of garlic oil was well tolerated by rats in the fed state. Also, 24 hr fasted rats could tolerate this dose of garlic oil, provided they were previously adapted to garlic oil feeding.