Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.     

Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells

The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution.     

Reciprocal activation of hypopharyngeal muscles and their effect on upper airway area.

We examined in awake goats, 1) with intact upper airways (UAW), the effect of altering chemical drive on pharyngeal constrictors [thyropharyngeus (TP) and hypopharyngeus (HP)] and a dilator [stylopharyngeus (SP)], and 2) with an isolated UAW, the effect of activation of these muscles on supraglottic UAW (UAW(SG)) area. During eupnea in nine goats with intact UAW, the TP and HP were active during expiration, whereas the SP exhibited tonic expiratory and phasic inspiratory activity. After mechanically induced apneas (MIA), TP activity increased (263%, P < 0.02), HP activity exhibited a small, varied response, and SP activity greatly decreased (10%, P < 0.02). During resumption of respiratory effort, all goats exhibited absent/reduced airflow, and when diaphragm activity was 95% of control, TP activity remained elevated (135%) and SP activity was reduced (56%, P < 0.02). During hypercapnia, 1) TP activity decreased (P < 0.02), 2) HP response varied, and 3) SP activity increased (P < 0.02). After MIA in six goats with isolated UAW, TP activity increased 198% (P < 0.02) and UAW(SG) area (endoscopically determined) decreased (to 15% of control, P < 0.02). During recovery from MIA, a correlation was found between UAW(SG) area and the ratio of SP to TP activity. We conclude that the reciprocal activation of mechanically opposing dilator and constrictor muscles in the hypopharynx is correlated to changes in the UAW(SG) area, and an imbalance in activity of these opposing muscles can lead to UAW(SG) narrowing.     

Angiogenic factor thymidine phosphorylase increases cancer cell invasion activity in patients with gastric adenocarcinoma

We investigated the biological role of thymidine phosphorylase (TP), an angiogenic factor, in gastric cancer cell migration and invasion and explored a therapeutic approach for high TP-expressing tumors using TP enzymatic inhibitor (TPI) and rapamycin. We established TP cDNA overexpressing gastric cancer cell lines (MKN-45/TP and YCC-3/TP) and did invasion and adhesion assays with Matrigel-coated transwell membranes. The related signal pathway using recombinant human TP (rhTP), deoxy-d-ribose (D-dRib), and signal pathway inhibitors (wortmannin, LY294002, and rapamycin) was investigated. First, AGS and MKN-1 gastric cancer cell lines showed dose-dependent up-regulation of invasiveness through Matrigel following treatment with rhTP or D-dRib. TP-overexpressing cancer cell lines displayed increased migration and invasion activity, which doubled with rhTP and D-dRib treatment. This activity depended on the enzymatic activity of TP, and TP stimulated the adhesion of cancer cells onto Matrigel and induced actin filament remodeling. Finally, we showed that this activity is related to increased phosphatidylinositol 3-kinase activity in TP-overexpressing cells and that combination treatment with rapamycin and TP enzymatic inhibitor produces an additive effect to abrogate TP-induced invasion. Taken together, TP increases the migration and invasion of gastric cancer cells, especially in TP-expressing cells. Therapies targeting TP might diminish the propensity for invasion and metastasis in gastric cancer.     

Thymidine phosphorylase inhibits apoptosis induced by cisplatin

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.     

Synthesis of [I-NaI3]] thymopoietin II and examination of its immunological effect on the uremic T-lymphocytes

A TP II analogue, [1-Nal3] TP II, was synthesized by a conventional solution method, followed by deprotection with 1M TFMSA-thioanisole (molar ratio 1:1) in TFA in the presence of Me2Se and m-cresol as scavengers. The synthetic [1-Nal3] TP II, TP II and [Phe (4 F)3] TP II were tested for comparative effect on the impaired T-lymphocyte transformation by PHA in uremic patients suffering from recurrent infectious diseases. The synthetic analogue was found to have stronger restorative activity than those of our synthetic TP II and [Phe (4F)3] TP II.     

Modulation of antinociceptive responses following tail pinch stress

Mild, non-noxious, oscillating pinches to a rat's tail elicits hyperphagia. The present study examined whether tail-pinch (TP) would exert hyperalgesic and hyperactive effects in rats that also exhibit the overeating response. The first experiment assessed TP effects upon reactivity to electric shock as measured by flinch-jump thresholds. Significant decreases in jump thresholds were observed 0 and 15, but not 30, min following TP. This effect persisted regardless of whether food was present or absent during TP. The second experiment assessed TP effects upon reactivity to heat as measured by hot-plate latencies. In contrast to jump thresholds, the shortened hot-plate latencies observed following TP persisted into the recovery period. In examining TP effects upon activity levels (Experiment 3), it was found that animals display similar patterns of temporally-declining activity regardless of whether TP was administered or not. Finally, TP selectively decreased the analgesic responses to two different doses of morphine and two different cold-water swim temperatures (Experiment 4). The TP-induced reductions occurred when TP was administered either before or after the analgesic manipulation. These data are discussed in terms of the nociceptive selectivity of the TP effect, and its influences upon analgesic processes.     

The anti-platelet drug ticlopidine inhibits FapC fibrillation and biofilm production: Highlighting its antibiotic activity

Multidrug resistance of bacteria and persistent infections related to biofilms, as well as the low availability of new antibacterial drugs, make it urgent to develop new antibiotics. Here, we evaluate the antibacterial and anti-biofilm properties of ticlopidine (TP), an anti-platelet aggregation drug, TP showed antibacterial activity against both gram-positive (MRSA) and gram-negative (E. coli, and P. aeruginosa) bacteria over a long treatment period. TP significantly reduced the survival of gram-negative bacteria in human blood though impact on gram-positives was more limited. TP may cause death in MRSA by inhibiting staphyloxanthin pigment synthesis, leading to oxidative stress, while scanning electron microscopy imaging indicate a loss of membrane integrity, damage, and consequent death due to lysis in gram-negative bacteria. TP showed good anti-biofilm activity against P. aeruginosa and MRSA, and a stronger biofilm degradation activity on P. aeruginosa compared to MRSA. Measuring fluorescence of the amyloid-reporter Thioflavin T (ThT) in biofilm implicated inhibition of amyloid formation as part of TP activity. This was confirmed by assays on the purified protein in P. aeruginosa, FapC, whose fibrillation kinetics was inhibited by TP. TP prolonged the lag phase of aggregation and reduced the subsequent growth rate and prolonging the lag phase to very long times provides ample opportunity to exert TP's antibacterial effect. We conclude that TP shows activity as an antibiotic against both gram-positive and gram-negative bacteria thanks to a broad range of activities, targeting bacterial metabolic processes, cellular structures and the biofilm matrix.     

Modulation of Brain Glutathione Reductase and Peroxiredoxin 2 by α-Tocopheryl Phosphate

α-Tocopheryl phosphate (αTP) is a phosphorylated form of α-tocopherol. Since it is phosphorylated in the hydroxyl group that is essential for the antioxidant property of α-tocopherol, we hypothesized that αTP would modulate the antioxidant system, rather than being an antioxidant agent per se. α-TP demonstrated antioxidant activity in vitro against iron-induced oxidative stress in a mitochondria-enriched fraction preparation treated with 30 or 100 µM α-TP. However, this effect was not observed ex vivo with mitochondrial-enriched fraction from mice treated with an intracerebroventricular injection of 0.1 or 1 nmol/site of αTP. Two days after treatment (1 nmol/site αTP), peroxiredoxin 2 (Prx2) and glutathione reductase (GR) expression and GR activity were decreased in cerebral cortex and hippocampus. Glutathione content, glutathione peroxidase, and thioredoxin reductase activities were not affected by αTP. In conclusion, the persistent decrease in GR and Prx2 protein content is the first report of an in vivo effect of αTP on protein expression in the mouse brain, potentially associated to a novel and biologically relevant function of this naturally occurring compound.     

Effect of antibiotic and vaccine therapies on the succinate dehydrogenase and phosphatase activity of liver cells of mice infected with Staphylococcus

Changes in the activity of succinate dehydrogenase (SDH), total and acid phosphatase (TP and AP) were studied in treatment of laboratory animals with rifampicin, lincomycin and with inactivated staphylococcal vaccine used alone or in combinations. It was shown that immunization of the animals with inactivated staphylococcal vaccine under conditions of experimental staphylococcal infection promoted stimulation of the enzyme activity. Rifampicin and lincomycin used for the treatment of such animals lowered the activity of the enzymes. The suppressing effect of the antibiotics increased with an increase in the period of their use. It should be noted that the inhibitory effect of rifampicin on the activity of SDH, TP and AP was less pronounced than that of lincomycin. The combined use of the vaccine and antibiotics for the treatment of the animals promoted an increase in the enzyme activity as compared to the use of the antibiotics alone. Sometimes the activity of SDH, TP and AP reached the control levels in such animals or the levels observed in the animals treated with the vaccine alone. Stimulation of the enzyme activity was more pronounced when the vaccine was used in combination with rifampicin.