Decreased Alkaline Phosphatase Activity Research Articles

The calcium channel Orai1 is required for osteoblast development: Studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1.

The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1fl/fl) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1fl/fl-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1fl/fl-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1fl/fl-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1fl/fl-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca2+ entry, exhibited greatly decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.

Effects of ultrasound-induced stress on gut microbiota of mice.

Prolonged stress causes deleterious effects on both the organism and its microbiota. In this study, we examined the effects of exposure to variable frequency ultrasound (US) on the gut microbiota-liver-brain axis of mice. This study was conducted on 20 mature clinically healthy sexually naive C57BL/6J male mice (42-45 days old). Group 1 (Normal) consisted of healthy intact mice (n = 10). Group 2 (Stress) consisted of mice subjected to US-induced stress (n = 10) for 20 days with alternating frequencies (20-45 kHz). Stool samples were collected on days 0, 10, and 20, and the corresponding DNA was later subjected to 16SrRNA sequencing. After mice were sacrificed on day 21, the leukocyte count, blood serum biochemical parameters, and liver and brain antioxidant status were measured. Behavioral testing was performed on days 17, 18, and 19. Ultrasound lead to higher stress and anxiety levels; increase in creatinine by 8.29% and gamma-glutamyltransferase activity by 5 times, a decrease in alkaline phosphatase activity by 38.23%, increase of de Ritis coefficient by 21.34%; increased liver and brain superoxide dismutase level by 20.8% and 21.5%, respectively; the stress-related changes in the gut microbiota composition - Bacteroidaceae and Firmicutes. Subjecting mice to 20 days of US-induced stress leads to systemic disorders due to oxidative stress and a decrease in the diversity of the gut microbiota.

Novel O-benzylcinnamic acid derivative L26 treats acute lung injury in mice by MD-2

Acute lung injury (ALI) is an inflammation-mediated respiratory disease that is associated with a high mortality rate. In this study, a series of novel O-benzylcinnamic acid derivatives were designed and synthesized using cinnamic acid as the lead compound. We tested the preliminary anti-inflammatory activity of the compounds by evaluating their effect on inhibiting the activity of alkaline phosphatase (ALP) in Hek-Blue-TLR4 cells, in which compound L26 showed the best activity and 7-fold more active than CIN. ELISA, immunoprecipitation, and molecular docking indicated that L26 targeted MD-2 protein and competed with LPS to bind to MD-2, which resulted in the inhibition of inflammation. In the LPS-induced mouse model of ALI, L26 was found to decrease ALP activity and inflammatory cytokine TNF-α release to reduce lung injury by inhibiting the NF-κB signaling pathway. Acute toxicity experiments showed that high doses of L26 did not cause adverse reactions in mice, and it was safe in vivo. Also, the preliminary pharmacokinetic parameters of L26 were investigated in SD rats (T1/2 = 4.246 h). In summary, L26 exhibited optimal pharmacodynamic and pharmacokinetic characteristics, which suggested that L26 could serve as a potential agent for the development of ALI treatment.

Evaluation of Levels of Phosphatases in the Lindane Exposed Fish, Channa punctatus

The alterations in the activities of acid and alkaline phosphatase are good indicators of stress due to the exposure of fish to any toxicant. The perturbations in their activities may result in the decreased immune responses and intestinal microflora activity. In the present study, the effect of sublethal concentrations of lindane (0.025, 0.05 and 0.1 mg/l) in different tissues of C. punctatus exposed for 96h has been studied. The data showed decrease in the acid phosphatase (AcP) activity in the following order: gills>muscle>heart>brain>kidney>liver, respectively, and for the Alkaline phosphatase (ALP) the order was gills>liver>muscle> heart>brain>kidney, respectively, for 96h treatment. At highest concentration of lindane (0.1 mg/l) tested; the maximum decrease in AcP activity was recorded in gills (47.11%) and minimum in liver (15.02%). The other organs such as muscle, heart, brain and kidney exhibited 38.12, 30.85, 29.97 and 16.85% decrease, respectively. The activity of ALP showed maximum decrease in gills (27.93%) and minimum in kidney (0.98%). At highest concentration of lindane (0.1 mg/l) tested, the brain, heart, liver and muscle registered 1, 6.29, 18.84 and 14.25% decrease in ALP activity, respectively, under this condition.

Impact of Paraquat on Acid and Alkaline Phosphatase Activity in Indian Major Carp <i>Cirrhinus mrigala</i> Hamilton

Paraquat is a non-selective aquatic herbicide used to control nuisance vegetation in aquatic bodies. It potentially causes fish toxicity. The present investigation was undertaken to evaluate alterations in Acid Phosphatase (ACP) and Alkaline Phosphatase (ALP) activity in fingerlings of commercially important Indian Major Carp Cirrhinus mrigala. Both ACP and ALP are popular biomarkers of pesticide toxicity- induced aquatic pollution. 96 hours of acute exposure to LC<sub>0</sub> and LC<sub>50</sub> concentrations and 30 days of chronic exposure to LC<sub>1/20th</sub> and LC<sub>1/10th</sub> concentration of paraquat were studied during the present experiment in selected four tissues viz. gill, liver, muscle and brain to interpret its comparative impact at different effective concentrations on selected target tissues of fish. 30 days of chronic exposure to LC<sub>1/10th</sub> dose of paraquat showed exceptionally highly significant (p<0.001) decline in ACP activity of all four tissues among all other treatment groups when compared with the control. The liver tissue of fishes exposed to both the acute concentrations of paraquat shows an exceptionally highly significant (p<0.001) decrease in ALP activity among all other treatment groups and tissues when compared with control. From the present study, it can be concluded that paraquat-induced decline in ACP and ALP activity in Cirrhinus mrigala can be utilized as a prominent biomarker of aquatic toxicity induced due to pesticides in non-targeted organisms like fish. Paraquat can be potentially considered an aquatic ecosystem hazard leading to severe environmental pollution and eco-toxicity.

Iodine-125 brachytherapy suppresses tumor growth and alters bone metabolism in a H1299 xenograft mouse model.

The present study aimed to investigate the efficacy of Iodine-125 (I-125) brachytherapy in a mouse model of non-small cell lung cancer, to further explore the efficacy and appropriate method of implantation of the I-125 radioactive seed. This study also aimed to determine the impact of brachytherapy on bone metabolism. A total of 18 mice were used to establish H1299 xenograft models, and were randomly assigned to three groups. These included non-radioactive seed implantation (Sham IM), fractionated I-125 seed implantation (Fractionated IM) and single I-125 seed implantation (Single IM) groups. Mice were euthanized after 28days of implantation. H&E staining, Ki67 immunohistochemistry, CD31 morphometric analysis and TUNEL immunofluorescence assays were respectively used to determine the histopathological changes, proliferation, micro-angiogenesis and apoptosis of tumors. In addition, bone volume and microstructure were evaluated using trabecular bone area (Tb.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N) and cortical thickness. Bone metabolic status was analyzed using histomorphometric staining of tartrate-resistant acid phosphate (TRAP) and alkaline phosphatase (ALP) expression in the femur, and using an ELISA assay to determine the expression of C-telopeptide of type 1 collagen (CTX-1) and procollagen type 1 n-terminal propeptide (P1NP) in the serum. Moreover, reverse transcription-quantitative PCR and western blotting were carried out for the analysis of bone remodeling-related gene expression in the bone tissue. Results of the present study demonstrated that compared with the Sham IM group, both the I-125 seed implantation groups, including Fractionated IM and Single IM, demonstrated significant therapeutic effects in both tumor volume and weight. More specifically, the most significant therapeutic effects on tumor inhibition were observed in the Fractionated IM group. Results of Ki67 and CD31 immunohistochemical staining suggested a notable reduction in tumor cell proliferation and micro-angiogenesis, and results of the TUNEL assay demonstrated an increase in tumor cell apoptosis. Although the cortical bone appeared thinner and more fragile in both I-125 seed implantation groups, no notable adverse changes in the morphology of the cancellous bone were observed, and the index of Tb.Ar, Tb.Th and Tb.n was not significantly different among Sham IM and I-125 implantation groups. However, alterations in bone metabolism were characterized by a decrease in CTX-1 and P1NP expression, accompanied by an increase in TRAP activity and a decrease in ALP activity. Results of the present study also demonstrated the notable suppression of osteocalcin and runt-related transcription factor 2. I-125 seed implantation may be an effective and safe antitumor strategy. Moreover, the use of fractionated implantation patterns based on tumor shape exhibited improved therapeutic effect on tumor suppression when the total number of I-125 seeds was equivalent along with reduced complications associated with bone loss.

SOX2 suppresses osteoblast differentiation of MC3T3-E1 cells through activating the transcription of LGR4.

Osteogenic differentiation is a crucial process of new bone formation. This study aimed to explore the roles and mechanism of SRY-Box Transcription Factor 2 (SOX2) on proliferation and osteogenic differentiation of MC3T3-E1 cells. Bone morphogenetic protein 2 (BMP2) was used to induce the osteogenic differentiation of MC3T3-E1 cells. The expression of SOX2 was determined by quantitative real-time PCR (RT-PCR) at different time points after induction. The SOX2 overexpression plasmids were constructed and transfected into MC3T3-E1 cells. Osteogenic differentiation was evaluated by Alizarin Red S staining and alkaline phosphatase (ALP) assay. The expressions of osteogenic differentiation markers including runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) were detected by western blot assay. Luciferase reporter and CHIP assays were used to confirm that SOX2 regulated the transcriptional activation of leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4). We found that SOX2 was down-regulated upon BMP2-induced osteogenic differentiation in MC3T3-E1 cells. Overexpression of SOX2 effectively inhibited osteogenic differentiation with decreased ALP activity, calcification, and osteogenic differentiation markers' expression including Runx2, OPN, and OCN. LGR4 was identified as a target of SOX2, and the inhibitory effect of SOX2 on osteogenic differentiation was reversed by knockdown of LGR4. The present study confirmed that SOX2 suppressed osteogenic differentiation of MC3T3-E1 cells through targeting LGR4, which possesses a therapeutic strategy for bone formation and generation.

Coastal wetland conversion to aquaculture pond reduced soil P availability by altering P fractions, phosphatase activity, and associated microbial properties

Reclamation and conversion of wetlands strongly affect nutrient cycling and ecosystem functions, while little attention has been paid to the effects of converting coastal wetland to aquaculture on the cycling and balance of soil phosphorus (P). Herein, we investigated soil P fractions, alkaline phosphatase (ALP) activity, and associated microbial properties following coastal wetland conversion in subtropical China. Soil P availability (especially resin-P) concentration and ALP activity in wetland were significantly higher than those in pond. The conversion of coastal wetlands to aquaculture significantly reduced the abundance and diversity of bacterial phoD genes and altered their community structure. The lower phosphatase activity and associated microbial properties after wetland conversion suggested a weaker capacity of microbes to transform organic P (Po) to inorganic P (Pi), consistent with the low P availability but the high Po:Pi ratio in pond. Structural equation modeling indicated that the conversion of the wetland to the pond decreased ALP activity and P availability by affecting soil variables such as bulk density, pH, the carbon: nitrogen ratio, and/or moisture. It was concluded that wetland conversion to pond reduced soil P availability and phosphatase activity, altered the abundance, diversity and community composition of the phoD gene, and ultimately affected coastal P cycles and balances. Moreover, an extended corollary is that the smaller amounts of variation in soil total P and lower labile P concentrations in pond than in wetland suggest that large amounts of P (introduced in feed and not harvested in shrimp) are “lost” from the system. Thus, aquaculture ponds might serve as a source of P for the surrounding environment. More investigations focusing on the P biogeochemical cycle and its potential impacts on adjacent ocean environments at regional and global scales is urgently needed, which could contribute to better management of coastal land uses.

Manzamine-A Alters In Vitro Calvarial Osteoblast Function.

Manzamine-A is a marine-derived alkaloid which has anti-viral and anti-proliferative properties and is currently being investigated for its efficacy in the treatment of certain viruses (malaria, herpes, HIV-1) and cancers (breast, cervical, colorectal). Manzamine-A has been found to exert effects via modulation of SIX1 gene expression, a gene critical to craniofacial development via the WNT, NOTCH, and PI3K/AKT pathways. To date little work has focused on Manzamine-A and how its use may affect bone. We hypothesize that Manzamine-A, through SIX1, alters bone cell activity. Here, we assessed the effects of Manzamine-A on cells that are responsible for the generation of bone, pre-osteoblasts and osteoblasts. PCR, qrtPCR, MTS cell viability, Caspase 3/7, and functional assays were used to test the effects of Manzamine-A on these cells. Our data suggests Six1 is highly expressed in osteoblasts and their progenitors. Further, osteoblast progenitors and osteoblasts exhibit great sensitivity to Manzamine-A treatment exhibited by a significant decrease in cell viability, increase in cellular apoptosis, and decrease in alkaline phosphatase activity. In silico binding experiment showed that manzamine A potential as an inhibitor of cell proliferation and survival proteins, i.e., Iκb, JAK2, AKT, PKC, FAK, and Bcl-2. Overall, our data suggests Manzamine-A may have great effects on bone health overall and may disrupt skeletal development, homeostasis, and repair.

Migration and toxicity of toltrazuril and its main metabolites in the environment.

Veterinary drugs heavily used in livestock are passed into the environment through different ways, resulting in risks to terrestrial environments and humans. The migration of toltrazuril (TOL), an important anticoccidial drug used intensively in livestock, and its main metabolites between the chicken manure compost, soil and vegetables was investigated, and then the impacts of TOL on the soil bacterial community and ARGs, soil enzyme activities and phytotoxicity were detected. In the process of aerobic composting for 80 days, except for toltrazuril sulfoxide (the degradation half-life was 59.74d), TOL and ponazuril (PON) were not significantly degraded. However, TOL and its metabolites were significantly degraded in fertilized soil, and the degradation half-life was 28.17-346.50d. Among the three drugs, only PON could migrate from soil to vegetables, and the residual concentrations of PON in lettuce and radish were 2.64-70.02μgkg-1 and 0-2.80μgkg-1, respectively. Moreover, TOL and its main metabolisms had no significant effect on the bacterial community structure and the abundance of antibiotic resistance genes during composting, but affected the microbial activity in the soil. The presence of TOL and its main metabolites reduced soil urease activity, increased catalase activity, and decreased alkaline phosphatase activity at the beginning and then increased slightly. They had negative effects on plant growth. Compared with the control group, the inhibition rates of TOL and its metabolites on lettuce and radish seed germination were 8.33% and 26.74% respectively, and the inhibition rates of root elongation length were 25.88% and 34.45% respectively. These results showed that TOL and its main metabolites were ineffectively removed by aerobic composting, and could be migrated from composting to soil and vegetables, which had adverse effects on soil enzyme activity and plant growth. Therefore, its environmental ecological risk and human health risk needs to be further evaluated.

Modulation of osteoblast differentiation and function by the P2X4 receptor

Bone cells are known to express multiple P2 receptor subtypes, and the functional effects of receptor activation have been described for many of these. One exception is the P2X4 receptor, which despite strong expression in osteoblasts and osteoclasts, has no defined functional activity. This study used the selective P2X4 receptor antagonists, 5-BDBD and PSB-12062, to investigate the role of this receptor in bone. Both antagonists (≥ 0.1 μM) dose-dependently decreased bone formation by 60–100%. This was accompanied by a ≤ 70% decrease in alkaline phosphatase activity, a ≤ 40% reduction in cell number, and a ≤ 80% increase in the number of adipocytes present in the culture. The analysis of gene expression showed that levels of osteoblast marker genes (e.g. Alpl, Bglap) were decreased in 5-BDBD treated cells. Conversely, expression of the adipogenic transcription factor PPARG was increased 10-fold. In osteoclasts, high doses of both antagonists were associated with a reduction in osteoclast formation and resorptive activity by ≤ 95% and ≤ 90%, respectively. Taken together, these data suggest that the P2X4 receptor plays a role in modulating bone cell function. In particular, it appears to influence osteoblast differentiation favouring the osteogenic lineage over the adipogenic lineage.

EPLINβ Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINβ. In this study, we investigated the role of EPLINβ in osteoblasts using EPLINβ-deficient (EPLINβGT/GT) mice. The skeletal phenotype of EPLINβGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINβGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINβGT/GT mice, suggesting aberrant cell adhesion. In EPLINβGT/GT osteoblasts, α- and β-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINβ knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINβ knockdown cells, suggesting an important role for EPLINβ in osteoblast formation. In conclusion, we propose that EPLINβ is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

Effect of miR-144-3p-Targeted Regulation of PTEN on Proliferation, Apoptosis, and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells under Stretch.

Objective To investigate the effects of miR-144-3p-targeted regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene on proliferation, apoptosis, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under retraction force. Methods The BMSCs of rats were randomly divided into the tension MSC group with detrusor stimulation and the MSC group without detrusor stimulation, after which osteogenic differentiation of BMSCs was induced in both groups. Alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the osteogenic differentiation ability of the two groups of cells. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression of miR-144-3p and PTEN in the two groups of cells after osteogenic differentiation. Bioinformatics website and dual luciferase reporter were used to detect the relationship between miR-144-3p and PTEN. The tension MSC group was used as a control group, and miR-144-3p mimics (miR-144-3p mimic group), mimic controls (mimic-NC group), PTEN interferers (si-PTEN group), and interference controls (si-NC group) were transfected into BMSCs. The BMSCs were then continuously stimulated for 24 h using a Flexercell in vitro cellular mechanics loading device, applying a draft force at a frequency of 1 Hz and a deformation rate of 18%. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) colorimetric assay; the expression levels of cyclin, cyclin-dependent kinases (CDK), BCL2-associated X (BAX), B-cell lymphoma-2 (BCL-2), and other cell cycle and apoptosis related proteins were detected by western blot (WB); and the osteogenic differentiation ability of MSC cells was detected by ALP staining and alizarin red staining. Results Compared with the MSC group, the level of miR-144-3p was significantly lower and the level of PTEN was significantly higher in the tension MSC group. ALP staining showed normal activity in the MSC group and decreased ALP activity in the tension MSC group compared to the MSC group. Alizarin red staining in the MSC group showed scattered calcium nodule formation, and alizarin red staining showed red nodules with a more uniform color distribution. Compared to the MSC group, the tension MSC group showed fewer, smaller, and lighter staining mineralized nodules. Compared with the tension group and mimic-NC group (si-NC group), the proliferation rate of cells in the miR-144-3p mimic group (si-PTEN group) was significantly higher; the expression levels of PTEN and BAX were significantly lower; and the expression levels of cyclin, CDK, and BCL-2 protein were significantly higher. ALP staining results revealed that the miR-144-3p mimic group (si-PTEN group) showed significantly higher osteogenic differentiation ability and ALP activity of MSC than the tension group and mimic-NC group (si-NC group). Conclusion miR-144-3p may inhibit apoptosis and promote proliferation and osteogenic differentiation of BMSCs under tension by targeting and regulating PTEN.

Investigating the effects of the marine drug Manzamine‐A related to craniofacial development in vitro

Manzamine‐A is a marine‐derived alkaloid which has anti‐viral and anti‐proliferative properties and is currently being investigated for its efficacy in the treatment of certain viruses (malaria, herpes, HIV‐1) and cancers (breast, cervical, colorectal). Manzamine‐A has been found to exert effects via modulation of SIX1 gene expression, a gene critical to craniofacial development via the WNT, NOTCH, and PI3K/AKTpathways. To date little work has focused on Manzamine‐A and how its use may effect craniofacial development. We hypothesize that Manzamine‐A, through SIX1, alters bone cell activity important to craniofacial skeletal development. As Manzamine‐A is a drug that has great potential in a variety of therapeutics, it is critical that we asses potential side effects that this drug could have on both patients and any developmental effects that may occur during use by expectant mothers. We aimed to assess the effects of Manzamine‐A on four cell types, pre‐osteoblasts, osteoblasts, pre‐osteoclasts, and osteoclasts. PCR, qrtPCR, MTS cell viability assays and Caspase 3/7 apoptosis assay were used to test the effects of Manzamine‐A on these cells. We also performed alkaline phosphatase (ALP) and Tartrate Resistant Acid Phosphatase (TRAP) assays to test the function of osteoblasts and osteoclasts respectively. To date we have found that Six‐1 is highly expressed in osteoblasts and their progenitors. In contrast Six‐1 has low expression levels in osteoclasts and their progenitors. Further, osteoblast progenitor cells exhibit great sensitivity to Manzamine‐A treatment exhibited by a significant decrease in cell viability, increase in cellular apoptosis, and decrease in ALP activity. Studies are ongoing for osteoclast lineage cells. Overall our preliminary data suggests Manzamine‐A may have great effects on bone health overall and may disrupt skeletal development if exposed during pregnancy or postnatal development.

Tolerance of juvenile Peruvian rock seabass (Paralabrax humeralis Valenciennes, 1828) and Peruvian grunt (Anisotremus scapularis Tschudi, 1846) to low-oxygen conditions.

Hypoxia is currently one of the greatest threats to coastal ecosystems worldwide, generating massive mortality of marine organisms, loss of benthic ecosystems and a decrease in fishery production. We evaluated and compared the tolerance to hypoxia of two species from different habitats of the Peruvian coast, the Peruvian rock seabass Paralabrax humeralis and the Peruvian grunt Anisotremus scapularis. The effect of hypoxia was measured as a function of the exposure time (progressive and chronic) on the behavioural and physiological responses of the two species, as well as on the enzymatic activity associated with the oxidative stress response of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and alkaline phosphatase (AKP). The ventilatory frequency was measured at two different temperatures (16 and 22°C) under progressive hypoxia conditions to determine the ventilatory critical point (Vcp). A. scapularis showed a higher Vcp than P. humeralis, which was positively affected by temperature. The median lethal time of A. scapularis was 36 min at 60% of oxygen saturation, while P. humeralis showed no mortality after 31 days of exposure at 5% oxygen saturation. Different enzymatic activity (P < 0.05) between species under hypoxia was recorded, in SOD (gill and muscle) and AKP (blood). A general tendency, under hypoxia, to slightly increase LDH activity (except for blood in A. scapularis, P< 0.05) and SOD activity (mainly in muscle of A. scapularis, P< 0.05), and decrease AKP activity (mainly in liver of P. humeralis, P< 0.05) was observed. The response of P. humeralis to hypoxia goes through a reduction in activity and metabolism, so this species can be considered hypoxia-tolerant, allowing it to face hypoxia events during prolonged periods. On the other hand, A. scapularis response to hypoxia prioritizes avoidance mechanisms and, together with other adaptations, makes it especially vulnerable to hypoxia and able to be considered hypoxia-intolerant.

Influence of chronic hyperhomocysteinemia on the features of bone metabolism in the case of lipopolysaccharide-induced periodontitis.

Periodontal disease is the second most common oral health problem after dental caries. This increasing prevalence makes it not only a health problem, but also a social issue. The pathogenesis of periodontal disease is associated with a number of adverse exogenous and endogenous factors, including hyperhomocysteinemia (HHcy). This study aimed to determine the features of bone metabolism in rats with lipopolysaccharide (LPS)-induced periodontitis combined with chronic thiolactone HHcy. Forty-eight white, non-linear, mature rats were divided into 4 groups: control (n = 12); LPS‑induced periodontitis (n = 12); chronic thiolactone HHcy (n = 12); and periodontitis combined with HHcy (n = 12). The rats were sacrificed the day after the last LPS injection or the day after the last homocysteine (Hcy) thiolactone administration. Bone metabolism was determined based on the activity of alkaline phosphatase (ALP) and acid phosphatase (AP) in blood serum and periodontal homogenate. A decrease in ALP activity (by 40.1%; р = 0.001) and the mineralization index (MI) (3.5 times; р < 0.001) with an increase in AP activity (2.0 times; р < 0.001) was observed in the periodontal homogenate of rats with LPS‑induced periodontitis. In the case of LPS‑induced periodontitis combined with chronic thiolactone HHcy, more pronounced changes in the activity of phosphatases and in MI were established as compared to rats with LPS‑induced periodontitis only. Chronic thiolactone HHcy enhances disturbances in bone metabolism in LPS‑induced periodontitis. The osteotoxic effect of HHcy is associated with the activation of osteoclastogenesis and enhanced bone resorption. However, further research is required on the subject.

Docosahexaenoic acid improves altered mineralization proteins, the decreased quality of hydroxyapatite crystals and suppresses oxidative stress induced by high glucose.

Patients with type 2 diabetes mellitus (DM2) experience an increased risk of fractures and a variety of bone pathologies, such as osteoporosis. The suggested mechanisms of increased fracture risk in DM2 include chronic hyperglycaemia, which provokes oxidative stress, alters bone matrix, and decreases the quality of hydroxyapatite crystals. Docosahexaenoic acid (DHA), an omega-3 fatty acid, can increase bone formation, reduce bone loss, and it possesses antioxidant/anti-inflammatory properties. The present study aimed to determine the effect of DHA on altered osteoblast mineralisation and increased reactive oxygen species (ROS) induced by high glucose concentrations. A human osteoblast cell line was treated with 5.5 mM glucose (NG) or 24 mM glucose (HG), alone or in combination with 10 or 20 µM DHA. The collagen type 1 (Col1) scaffold, the expression of osteocalcin (OCN) and bone sialoprotein type-II (BSP-II), the alkaline phosphatase (ALP) specific activity, the mineral quality, the production of ROS and the mRNA expression of nuclear factor erythroid 2-related factor-2 (NRF2) were analysed. Osteoblasts cultured in HG and treated with either DHA concentration displayed an improved distribution of the Col1 scaffold, increased OCN and BSP-II expression, increased NRF2 mRNA, decreased ALP activity, carbonate substitution and reduced ROS production compared with osteoblasts cultured in HG alone. DHA counteracts the adverse effects of HG on bone mineral matrix quality and reduces oxidative stress, possibly by increasing the expression of NRF2.

Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation.

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.

Pathomorphological and histochemical parameters in the internal organs of carp infested Cariophyllaeus brachycollis

Pathomorphological and morphohistochemical changes were performed in the intestine of carp at the location of cysts of Cariophyllaeus brachycollis and other organs that do not have direct contact, primarily such as liver, kidneys, spleen, heart. A pathological autopsy of intensively infected carp with C. brachicollis cystism showed a slightly enlarged clay-yellowish liver; the gallbladder is often overflowing with bile. The kidneys are in most cases flabby and surrounded by a jelly-like mass, the spleen is also partially enlarged, and its edges are rounded, indicating dystrophic changes in these organs. The results of morphohistochemical studies have shown that granular dystrophy is observed in carp cells during cariophilic invasion in liver parenchyma cells. The content of α-amino acids and acid phosphatase activity in dystrophically altered hepatocytes increases significantly. The content of CHIC-positive substances increases in and around the veins and sinusoidal capillaries. These processes coincide with the destruction of individual liver beams. At a karyophilosis in kidneys of carp expansion of distal departments of tortuous tubules and a granular dystrophy is observed. In addition, a decrease in the content of neutral glycoproteins, alkaline phosphatase activity with a simultaneous increase in protein and acid phosphatase activity is observed in the brush border of nephrocytes. The appearance of pyroninophilic cells, decrease in the content of CHIC-positive substances, decrease in alkaline activity and increase in acid phosphatase activity were detected in the renal corpuscle. In the endothelium of blood vessels and capillaries, an increase in protein content, a decrease in alkaline phosphatase activity and an increase in acid phosphatase activity were found. Morphohistochemical studies in the capsule of the carp spleen indicate an increase in such indicators as pyroninophilia, CHIC-positive reaction, the presence of α-amino acids, acid phosphatase activity and decreased alkaline phosphatase activity. At the same time in the walls of blood vessels, there is an increase in the CHIC reaction and α-amino acids, a decrease in alkaline phosphatase, and an increase in acid phosphatase activity. Around the vessels in the reticular tissue of the spleen and between the cells of the pancreas significantly increases the accumulation of reticulocytes with increased acid phosphatase, bright pyroninophilic lymphocytes, and hemosiderocytes with an enhanced CHIC response. Morphohistochemical studies of the heart of fish are accompanied by dilation and overflow of blood cavities and vessels of the heart, small hemorrhages, and locally – pyknosis of muscle cell nuclei and granular dystrophy in the myocardium. At the same time in the endocardium – an increase in the number of lymphocytes and plasma cells with high content of DNA and RNA, as well as a marked increase in α-amino acids in connective tissue elastic fibers and squamous epithelial cells of the atrial and ventricular epicardium, alkaline and acid phosphatase. The amount of CHIC-positive substances is reduced in the striated muscle cells of the myocardium and the walls of the blood vessels of the epicardium. Thus, morphological and histochemical studies indicate profound and local changes in karyophilosis in the intestines of carp. In organs that are not directly related to the invasive onset, moderate histopathological and histochemical changes are observed, indicating a moderate severity of functional impairment.

Endoplasmic reticulum stress response mediated by the PERK-eIF2α-ATF4 pathway is involved in odontoblastic differentiation of human dental pulp cells

ObjectiveRNA-activated protein kinase-like ER-resident kinase (PERK) was a major transducer of Endoplasmic reticulum (ER) stress response and it directly phosphorylated α-subunit of eukaryotic initiation factor 2 (eIF2α), which specifically promoted the translation of activating transcription factor 4 (ATF4), an important transcription factor in cells’ differentiation. The purpose of this study was to establish whether ER stress mediated by PERK-eIF2α-ATF4 pathway was involved in odontoblastic differentiation of human dental pulp cells (DPCs). MethodsDPCs were isolated from extracted teeth and cultured in odontogenic medium. A recombinant lentiviral vector was constructed to transfect DPCs for PERK knockdown. Alkaline phosphatase (ALP) and Alizarin red S staining were used to characterize the odontoblastic differentiation. Real-time polymerase chain reactions (RT-PCR) were performed to analyze the genes’ expressions in DPCs’ odontoblastic differentiation. The mRNA and protein levels of ER stress markers were examined by RT-PCR and western blot. ResultsDPCs cultured in odontogenic media showed increased ALP activity and mineralized nodule formation. Notably, treatment with differentiation medium resulted in the up-regulation of genes, such as osteocalcin (OCN), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP), splicing x-box binding protein-1 (sXBP1), ATF4 and glucose-regulated protein 78 (GRP78). Meanwhile, the expressions of PERK-eIF2α-ATF4 pathway proteins, phosphorylated PERK, phosphorylated eIF2α and ATF4, increased in odontoblastic induction cells compared with controls. Furthermore, inhibition of PERK (PERK knockdown) decreased ALP activity and matrix mineralization in DPCs accompanied by the decrease expression of phosphorylated eIF2α and ATF4. ConclusionThese results suggested that PERK-eIF2α-ATF4 pathway was involved in the odontoblastic differentiation of DPCs.