Different effects of selenium on cyclic AMP metabolism in hepatoma cells and normal liver cells

Sodium selenite administered to normal and hepatoma (HA)-bearing mice sc (2 mg/kg) or ip (1 mg/kg) led to a significant increase in cyclic AMP (cAMP) level and a decrease in cAMP phosphodiesterase (PDE) activity in HA cells. In contrast, in tumor host liver and normal liver the cAMP level was reduced, and the PDE activity was slightly elevated, whereas the cAMP adenylate cyclase (AC) was little affected by Na₂SeO₃, if at all. These results imply that selenite-induced changes in PDE activity play a decisive role in regulating intracellular cAMP level. Kinetic studies revealed that there were different forms of PDE. TheK _m value of PDE isozymes in normal liver and host liver were identical, but differed from those of HA. It seems likely that the selective responsiveness of PDE to selenite may be related to the difference in PDE isozyme patterns. Addition of DBcAMP to the culture medium resulted in an inhibition of³H-thymidine incorporation into hepatoma cells, indicating that the inhibition of HA cell proliferation was cAMP-mediated. Thus, selenium has been shown to exert a selective effect on cAMP metabolism of hepatoma cells, which may account for its inhibitory effects on cancer cells.     

Synthesis and characterization of iodopropyl derivatives of adenosine 3′,5′-cyclic-monophosphate: Potential affinity labels of cAMP binding sites in enzymes

The syntheses of two potential cAMP affinity lables, 1,N ⁶-(3-iodopropyleno)adenosine 3′,5′-cyclic-monophosphate and 2′-O-(2-iodo-3-hydroxypropyl) adenosine 3′,5′-cyclic-monophosphate, by a two-step chemical procedure are described. TheN ⁶- and 2′-O-allyl intermediates were prepared selectively by alkylation of cAMP in organic and alkaline aqueous solutions, respectively. Treatment of theN ⁶-allyl derivative withN-iodosuccinimide resulted in iodine addition to the double bond and cyclization to theN ¹ position of the purine ring. The iodohydrin analog was synthesized by reaction of 2′-O-allyl-cAMP with potassium iodide and thallium trichloride in acetate buffered solution. The products were isolated by column chromatography and characterized by thin-layer chromatography, elemental analysis, and ultraviolet,¹³C, and¹H NMR spectroscopy. The cAMP analogs were found to react with lysine and cysteine. Both cAMP derivatives were tested for their reaction with the low-K _m cAMP phosphodiesterase of human platelets. The ribose-substituted analog functioned as a competitive inhibitor (K _I =0.72 μM) and caused a time-dependent irreversible inactivation of the phosphodiesterase. In contrast, the purine-substituted derivative acted neither as a reversible competitive inhibitor nor as an irreversible inactivator of the enzyme. These results indicate the specificity of these potential cAMP analogs in their interaction with the phosphodiesterase.     

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3′,5′-AMP for the utilization of various carbohydrates

Adenine requiring mutants of Serratia marcescens SM-6-F'lac ⁺ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5′AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5′AMP at a rate of 1.6–2.0 μmoles min⁻¹ mg⁻¹ protein. The loss of the phosphodiesterase activity in S. marcescens cpd W1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257^pc-1 of Escherichia coli. The induced synthesis of β-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd ⁺ strain. Starting from S. marcescens cpd W1181 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40–60% of that of the parental strain which is about 4×10⁻⁴ M. However, the adenylate cyclase in cell extracts of the mutants W1237 and W1270 was like that of the corresponding cya ⁺ strain (about 2×10⁻² μmoles min⁻¹ mg⁻¹ protein).     

Biochemical and cellular aspects of the anticancer activity of selenium

Aberrant differentiation is a frequent hallmark of tumors, suggesting that modulators for differentiation and proliferation play a role in multistage carcinogenesis and that their use can also be exploited in cancer chemoprevention and therapy. We have demonstrated that selenium (Se) may be a modulator for the differentiation and proliferation of tumor cells. Evidence has been obtained that Se exerts the following effects: reversing changes of biochemical phenotypes toward normal levels, including reduction of cGMP level and cAMP-dependent protein kinase isozyme type I; increase in cAMP level and cAMP-dependent protein kinase isozyme type II, and altering membrane properties. Furthermore, we have obtained support for this hypothesis utilizing experiments on cultured human liver cell lines. It is demonstrated that Se can lead to the following changes: a. reduction of mitotic index; b. increase in the adhesiveness of cells; c. decrease in confluent saturation density and induction of an early contact inhibition; and d. decrease in tumorigenicity. For the purpose of comparison, the effects of Se on the normal counterparts was also studied. Contrary to what was observed above, there was no significant change in both biochemical and cellular aspects of normal cells treated analogously.     

Epigenetic Enhancement of Brain-Derived Neurotrophic Factor Signaling Pathway Improves Cognitive Impairments Induced by Isoflurane Exposure in Aged Rats

Isoflurane-induced cognitive impairments are well documented in animal models; yet, the molecular mechanisms remain largely to be determined. In the present study, 22-month-old male Sprague-Dawley rats received 2 h of 1.5 % isoflurane or 100 % oxygen daily for 3 consecutive days. For the intervention study, the rats were intraperitoneally injected with 1.2 g/kg sodium butyrate 2 h before isoflurane exposure. Our data showed that repeated isoflurane exposure significantly decreased the freezing time to context and the freezing time to tone in the fear conditioning test, which was associated with upregulated histone deacetylase 2, reduced histone acetylation, and increased inflammation and apoptosis in the hippocampus, and impairments of brain-derived neurotrophic factor (BDNF)-tyrosine kinase receptor B (TrkB) and the downstream signaling pathway phospho-calmodulin-dependent protein kinase and phospho-cAMP response element-binding protein. These results suggest that isoflurane-induced cognitive impairments are associated with the declines in chromatin histone acetylation and the resulting downregulation of BDNF-TrkB signaling pathway. Moreover, the cognitive impairments and the signaling deficits can be rescued by histone deacetylase inhibitor sodium butyrate. Therefore, epigenetic enhancement of BDNF-TrkB signaling may be a promising strategy for reversing isoflurane-induced cognitive impairments.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Functional role of membrane-bound adenylyl cyclases and coupled to them receptors and G-proteins in regulation of fertility of spermatozoa

The cAMP-dependent signaling cascades play the key role in regulation of fertility of spermatozoa. Synthesis of cAMP in spermatozoa is realized both by soluble, and by transmembrane (membrane-bound) forms of adenylyl cyclases (AC). For the recent years numerous data appeared about the presence in spermatozoa at different stages of their maturation of a wide spectrum isoforms of membrane-bound AC and their regulation by hormones and hormone-like substances via the coupled to B-proteins receptors (GPCR). Agonists of GPCR in spermatozoa can be adenosine, biogenic amines, peptide hormones, odorants. Study of structural-functional organization and regulatory properties of AC of the signal system in spermatozoa is of great practical significance for reproductive technologies, as via the membrane-bound AC forms and signal cascades there are controlled such processes as motility and chemotaxis of spermatozoa, their capability for capacitation acrosomal reaction. In the review there are summarized and analyzed data on functioning and role of AC of signal system in spermatozoa of human and vertebrate animals and are discussed achievements and unsolved problems in this field.     

The selective roles of chaperone systems on over-expression of human-like collagen in recombinant Escherichia coli

Human-like collagen (HLC) is a novel biomedical material with promising applications. Usually, insoluble HLC was formed due to over-expression. In order to improve the production of soluble HLC, the effective chaperone proteins and their mediation roles on HLC were clarified. Trigger factor (TF) pathway with low specificity and high binding affinity to nascent chains could increase soluble HLC expression; GroEL-GroES could increase the expression level of HLC by assisting the correct folding of HLC and increase mRNA level of the gene coding for HLC by enhancing mRNA stability. DnaK chaperone system did not work positively on soluble HLC due to the unbalanced ratio of DnaK:DnaJ:GrpE, especially too high GrpE significantly inhibited DnaK-mediated refolding. The production of soluble HLC with co-expression of exogenous TF and GroEL-GroES was increased by 35.3 % in comparison with the highest value 0.26 g/L reported previously.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

Cell cycle arrest induced by theophylline in root meristem of Haplopappus gracilis

Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, induced a block of the cell cycle in G₁, a temporary arrest in G₂ and 70% decrease in the uptake of labelled thymidine in roots of Haplopappus. These effects are compared to those previously found with aminophylline and discussed in view of the possible involvement of cAMP in the regulation of the cell cycle in plants.     

EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts

Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.     

A prepared anti-MSTN polyclonal antibody reverses insulin resistance of diet-induced obese rats via regulation of PI3K/Akt/mTOR&FoxO1 signal pathways

Suppression of myostatin (MSTN) is associated with skeletal muscle atrophy and insulin resistance. However, the mechanisms by which MSTN regulates insulin resistance are not well known. We have explored the signaling pathways through which MSTN regulates insulin resistance in diet-induced obese rats using a polyclonal antibody for MSTN. The anti-MSTN polyclonal antibody significantly improved insulin resistance and whole-body insulin sensitivity, decreased MSTN protein expression in muscle samples by 39 % in diet-induced obese rats. Furthermore, the anti-MSTN polyclonal antibody significantly enhanced PI3K activity (140 %), Akt phosphorylation (86 %), GLUT4 protein expression (23 %), the phosphorylation of mTOR (21 %), and inhibited the phosphorylation of FoxO1 (57 %), but did not affect the phosphorylation of GSK-3β. Thus, suppression of MSTN by the anti-MSTN polyclonal antibody reverses insulin resistance of diet-induced obesity via MSTN/PI3K/Akt/mTOR and MSTN/PI3K/Akt/FoxO1 signaling pathways.     

Impregnation of the Bacterial Cellulose Membrane with Biologically Produced Silver Nanoparticles

Different wound dressings with antibacterial property have been surveyed and one among them is bacterial cellulose (BC). Since the BC does not have antibacterial property, the biologically produced silver nanoparticles (SNPs) were impregnated into the BC. For the BC production, Hestrin–Schramm broth was used. Formation of the BC was proven by enzymatic hydrolysis. For SNPs production, the bacterial supernatant was treated with AgNO₃ and formation of SNPs was monitored through spectrophotometer, TEM and XRD. For impregnation of SNPs into the BC, the cleaned membrane was placed in the bacterial supernatant that contained 1 mmol of AgNO₃. The antibacterial assay was done for the BC/SNPs. Enzymatic hydrolysis proved the presence of the BC. Spectrophotometer and XRD results showed the formation of SNPs. TEM analysis revealed the presence of SNPs with sizes around 5–100 nm. SEM micrographs showed the impregnation of SNPs into the BC. Antibacterial test exhibited the antibacterial activity of the BC/SNPs.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.