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Nisin, a known food preservative, has antimicrobial and anticancer properties. Nisin is a polycyclic polypeptide with five internal rings; a feature that strongly influence the choice of solvent to attain effectivity of the compound for all cell culture experiments. Previous studies used a number of solvents but comparison between these to identify the best solvent have not been reported. Aim: To investigate the effect of nisin ZP (NZP) when dissolved in four different solvents on the viability of MG63 osteosarcoma cells. Materials and methods: MG-63 cells were incubated overnight at 37°C, 5% CO2 and treated with increasing concentration of dimethyl sulfoxide (DMSO), methanol and acetic acid to observe the cytotoxic effects of these chemicals onto the cells via MTS assay. NZP was then dissolved in distilled water and the three solvents in increasing concentrations and applied to the cultured cells. Cell viability and morphology were documented. Results: Acetic acid, DMSO and methanol at 0.05% maintained more than 90% viability of MG-63. NZP precipitated at higher concentrations (≥ 800µg/ml) when dissolved in DMSO, methanol and distilled water which interfered with the measurement of formazan density in the cell viability assay. Increasing doses of NZP in 0.05% acetic acid showed the least interference with the desirable reduction of the cancer cell viability. Microscopically, as concentration of NZP increases, cell shrinkage and apoptotic bodies were more visible. In addition, brown precipitates were abundantly observed. Conclusion: Acetic acid provides the optimal solubility of NZP and the best solvent to use in experiments involving MG-63 cells.

The present work evaluated the protective effects of Spirulina against the bone fragility caused by Garcinia cambogia in high-fat diet induced obese rats. High-fat diet and high-fat emulsion (HFD+HFE) were administered via oral gavage to 30 six-week-old female Sprague Dawley rats for six weeks to induce obesity, except for a normal group (n = 6). Following four weeks of treatment, the diet-induced obese groups were orally administered, daily, with (1) G. cambogia (GC); (2) Spirulina (S); and (3) G. cambogia + Spirulina (GC+S). The normal and obese control groups were treated with equal volumes of 0.9% saline water. It was found that GC significantly decreased body mass index (BMI) below the obese range (0.68 g/cm2). Additionally, GC altered bone mineral density (BMD), increased phosphate and calcium levels, and decreased maximum force and mineral apposition rates (MAR) as compared to the obese control group (p < 0.05). Bone fragility caused by GC was confirmed by the decrease in bone formation marker osteocalcin (OCN), as well as an increase in bone resorption receptor activator of nuclear factor kappa-B ligand (RANKL) and tartrate-resistant acid phosphatase type 5b (TRAP5b) as compared to the obese control group. Spirulina also decreased the BMI of the obese rats. Spirulina also increased blood bone markers, BMD, maximum force, and Young’s modulus. Rats supplemented with GC+S demonstrated higher double-labelled surface (dLS/BS) and MAR as compared to those in the GC group (p < 0.05). Meanwhile, the S group demonstrated improvement in all dynamic histomorphometric indices. S and GC+S groups demonstrated bone formation upregulation and bone resorption downregulation, thus indicating a bone protective effect of Spirulina. Overall, GC treatment led to bone fragility. GC+S treatment significantly augmented bone formation and mineralisation in obese rats as compared to the GC treatment alone. Rats in the S group demonstrated effective weight reduction while showing no destructive effects on the bone.

Background: Diabetes Mellitus (DM) is characterized by hyperglycemia (high blood glucose levels) which is due to the destruction of insulin-producing β-cells in the islets of Langerhans in the pancreas. It is associated with oxidative and endoplasmic reticulum stress. The plant alkaloid Palmatine has been previously reported to possess antidiabetic and antioxidant properties as well as other protective properties against kidney and liver tissue damage. Objective: Here, we investigated the ability of Palmatine to reduce the up-regulation of chaperone proteins Glucose Regulatory Protein 78 (GRP78), and Calreticulin (CALR) protein in a Streptozotocin (STZ)-induced diabetic rat model. Methods: Streptozotocin (STZ) induced diabetes in Sprague Dawley rats treated with 2mg/kg of Palmatine for 12 weeks after the elevation of plasma glucose levels above 11mmol/L post-STZ administration. Proteins were extracted from the pancreas after treatment and Two-Dimensional gel electrophoresis (2-DE), PDQuest 2-D analysis software genomic solutions and mass spectrometer were used to analyze differentially expressed protein. Mass Spectrometry (MS/MS), Multidimensional Protein Identification Technology (MudPIT) was used for protein identification. Results: There was an up-regulation of the expression of chaperone proteins CALR and GRP78 and down-regulation of the expression of antioxidant and protection proteins peroxidoxin 4 (Prdx4), protein disulfide isomerase (PDIA2/3), Glutathione-S-Transferase (GSTs), and Serum Albumin (ALB) in non-diabetic rats. Palmatine treatment down-regulated the expression of chaperone proteins CALR and GRP78 and up-regulated the expression of Prdx4, PDIA2/3, GST, and ALB. Conclusion: Palmatine may have activated antioxidant proteins, which protected the cells against reactive oxygen species and endoplasmic stress. The result is in consonance with our previous report on Palmatine.

There has been a shift towards utilizing natural products as an adjunct therapy to standard treatment in the prevention of coronary artery disease, and Trigonella foenum graecum (TFG) is one of the potential natural products of interest. In the present study, we attempted to determine the effects of TFG and its saponins on atherosclerosis related biomarkers in vitro. Protein expression of markers of inflammation, endothelial activation and transcription factors were measured by Procarta™ and ELISA assays. Gene expression of the same markers were determined by qPCR and the interaction between monocytes and HCAECs were evaluated through monocyte binding assay following 16 h of treatment with TFG and saponins. Both TFG and its saponins exhibited reducing effects on atherosclerosis-related markers. Based on the area under the curve (AUC) analysis, TFG reduced protein and gene expressions of ICAM-1 and VCAM-1 better than the saponins, while saponins reduced E-selectin expression better than TFG. Saponins showed a reduction of gene and protein expressions of IL-6, IL-8, NF-κB p50 and p65 better than TFG. TFG is more effective in reducing binding of monocytes to endothelial cells than saponins. TFG better reduced endothelial activation but exerted weaker anti-inflammatory effects than saponins, suggesting the possible synergism with other compounds in the crude extract which enhances attenuation of endothelial activation while inhibiting anti-inflammatory properties of saponins in the crude extract. HIGHLIGHTS This research determined the effects of Trigonella foenum graecum and its saponins on atherosclerosis-related biomarkers in-vitro using human coronary artery endothelial cells Trigonella foenum graecum can reduce endothelial activation better than its saponins Saponins appears to exert better anti-inflammatory effects in its single form GRAPHICAL ABSTRACT

BACKGROUND: Hyperthermia is used as an adjuvant treatment to sensitize cancer cells to subsequent radiotheraphy or chemotherapy. The aim of this study was to study the effect of severe hyperthermia on osteosarcoma cells and its underlying causes.METHODS: Short-term (1 h) severe hyperthermia (45°C) was applied to osteoblast-like osteosarcoma cells (MG-63) and the heat shock response and cell death mechanisms were investigated after recovery at 37°C for 72 h.RESULTS: Cell viability was significantly reduced (p

Many studies reported that antioxidants and dietary supplements increase tumour progress and reduce survival. Hyperthermia is an adjuvant treatment to sensitise cancer cells for radio- or chemotherapy. The current study was carried out to determine the effects of berberine chloride and hyperthermia on bone cancer cells. MG-63 osteosarcoma cells were treated with hyperthermia (39, 43 and 45°C, respectively) for 1 h. Then, the cells were treated with a low toxic dose of berberine chloride (80 μg/mL). After that, all treated groups were recovered at 37°C for 24 h. Finally, all groups were treated with hyperthermia (39, 43 and 45°C) for the second time (1 h) and were recovered for 3 h at 37°C. Cells exposed to hyperthermia without treatment of berberine chloride were used as hyperthermia control. Cells treated with 80 μg/mL of berberine at 37°C served as berberine control and cells incubated at 37°C were used as an untreated control. All treated groups showed significant apoptosis compared to the control group (p

Increasing interest in vascular pseudo-ossification has alarmed the modern atherosclerotic society. High phosphate is one of the key factors in vascular pseudo ossification, also known as vascular calcification. The active process of deposition of the phosphate crystals in vascular tissues results in arterial stiffness. High phosphate condition is mainly observed in chronic kidney disease patients. However, prolonged exposure with high phosphate enriched foods such as canned drinks, dietary foods, etc. can be considered as modifiable risk factors for vascular complication in a population regardless of chronic kidney disease. High intake of vitamin K regulates the vascular calcification by exerting its anti-calcification effect. The changes in serum phosphate and vitamin K levels in a normal individual with high phosphate intake are not well investigated. This review summarised the underlying mechanisms of high phosphate induced vascular pseudo ossification such as vascular transdifferentiation, vascular apoptosis and phosphate uptake by sodium-dependent co-transporters. Pubmed, Science Direct, Scopus, ISI Web of Knowledge and Google Scholar were searched using the terms ‘vitamin K’, ‘vascular calcification, ‘phosphate’, ‘transdifferentiation’ and ‘vascular pseudoossification’. Vitamin K certainly activates the matrix GIA protein and inhibits vascular transition and apoptosis in vascular pseudo-ossification. The present view highlighted the possible therapeutic linkage between vitamin K and the disease. Understanding the role of vitamin K will be considered as potent prophylaxis agent against the vascular disease in near future.

Spirulina (blue-green algae) contains a wide range of nutrients with medicinal properties which include β-carotene, chromium, and moderate amounts of vitamins B12. This study aims to determine the preventive effect of spirulina against bone fragility linked to type 2 diabetes mellitus. Thirty Sprague-Dawley rats were divided into five groups (n = 6) and diabetes was induced using streptozocin. Rats with a plasma glucose level of 10 mmol/L and above were orally treated for twelve weeks with either a single dose of spirulina, metformin, or a combined dose of spirulina + metformin per day. After the treatment, blood and bones were taken for biochemical analysis, three-dimensional imaging, 3-point biomechanical analysis, histology imaging and gene expression using qPCR. Results showed that diabetes induction and treatment with metformin caused destruction in the trabecular microarchitecture of the femur bone, reduction in serum bone marker and expression of bone formation marker genes in the experimental rats. Spirulina supplementation showed improved trabecular microarchitecture with a denser trabecular network, increased 25-OH vitamin D levels, and lowered the level of phosphate and calcium in the serum. Biomechanical tests revealed increased maximum force, stress strain, young modulus and histology images showed improvement in regular mesh and an increase in osteoblasts and osteocytes. There was an increase in the expression of bone formation marker osteocalcin. The results suggest that spirulina supplementation was more effective at improving bone structural strength and stiffness in diabetic rats compared to metformin. Spirulina may be able to prevent T2DM-related brittle bone, lowering the risk of fracture.

Crocus sativus L. or saffron has been shown to have anti-atherogenic effects. However, its effects on key events in atherogenesis such as endothelial activation and monocyte-endothelial cell binding in lipolysaccharides (LPS)-stimulated in vitro model have not been extensively studied.ObjectivesTo investigate the effects of saffron and its bioactive derivative crocin on the gene and protein expressions of biomarkers of endothelial activation in LPS stimulated human coronary artery endothelial cells (HCAECs).MethodologyHCAECs were incubated with different concentrations of aqueous ethanolic extracts of saffron and crocin together with LPS. Protein and gene expressions of endothelial activation biomarkers were measured using ELISA and qRT-PCR, respectively. Adhesion of monocytes to HCAECs was detected by Rose Bengal staining. Methyl-thiazol-tetrazolium assay was carried out to assess cytotoxicity effects of saffron and crocin.ResultsSaffron and crocin up to 25.0 and 1.6 μg/ml respectively exhibited >85% cell viability. Saffron treatment reduced sICAM-1, sVCAM-1 and E-selectin proteins (concentrations: 3.13, 6.25, 12.5 and 25.0 μg/ml; 3.13, 12.5 and 25.0 μg/ml; 12.5 and 25.0, respectively) and gene expressions (concentration: 12.5 and 25.0μg/ml; 3.13, 6.25 and 25.0 μg/ml; 6.25, 12.5 25.0; respectively). Similarly, treatment with crocin reduced protein expressions of sICAM-1, sVCAM-1 and E-selectin (concentration: 0.2, 0.4, 0.8 and 1.6 μg/ml; 0.4, 0.8 and 1.6 μg/ml; 0.8 and 1.6 μg/ml; respectively] and gene expression (concentration: 0.8 and 1.6 μg/ml; 0.4, 0.8 and 1.6 μg/ml; and 1.6 μg/ml, respectively). Monocyte-endothelial cell interactions were reduced following saffron treatment at concentrations 6.3, 12.5 and 25.00 μg/ml. Similarly, crocin also suppressed cellular interactions at concentrations 0.04, 0.08, 1.60 μg/ml.ConclusionSaffron and crocin exhibits potent inhibitory action for endothelial activation and monocyte-endothelial cells interaction suggesting its potential anti-atherogenic properties.

Aims Phytoestrogens and xenoestrogens act as agonists/antagonists in bone formation and differentiation. Strong bones are depending of the ability of osteoblasts to form new tissue and to mineralize the newly formed tissue. Dysfunctional or loss of mineralization leads to weak bone and increased fracture risk. In this study, we reported the effect of different types of phytoestrogens (daidzein, genistein and equol) on mineralization in hFOB 1.19 cells stimulated with bisphenol A (BPA). Main methods Cell mineralization capacity of phytoestrogens was investigated by evaluating calcium, phosphate content and alkaline phosphatase activity. Bone related markers, osteocalcin and osteonectin, responsible in maintaining mineralization were also measured. Key findings BPA is significantly interfering with bone mineralization in hFOB 1.19 cells. However, the enhanced mineralization efficacy of daidzein and genistein (particularly at a dose of 5 and 40 μg/mL, respectively) was evidenced by increasing calcium and phosphate content, higher ALP activity, compared to the untreated BPA group. The quantitative analyses were confirmed through morphological findings. Osteocalcin and osteonectin levels were increased in phytoestrogens-treated cells. These findings revealed the potential effect of phytoestrogens in reverting the demineralization process due to BPA exposure in hFOB 1.19 cells. Significance We found that osteoblast differentiation and mineralization were maintained following treatment with phytoestrogens under BPA exposure.

Bisphenol A (BPA) (2,2,-bis (hydroxyphenyl) propane), a well-known endocrine disruptor (ED), is the exogenous chemical that mimic the natural endogenous hormone like oestrogen. Due to its extensive exposure to humans, BPA is considered to be a major toxicological agent for general population. Environmental exposure of BPA results in adverse health outcomes including bone loss. BPA disturbs the bone health by decreasing the plasma calcium level and inhibiting the calcitonin secretion. BPA also stimulated differentiation and induced apoptosis in human osteoblasts and osteoclasts. However, little is known about the underlying mechanisms of the untoward effect of BPA against bone metabolism. The present review gives an overview on the possible mechanisms of BPA towards bone loss. The previous literature shows that BPA exerts its toxic effect on bone cells by binding to the oestrogen related receptor-gamma (ERγ), reducing the bone morphogenic protein-2 (BMP-2) and alkaline phosphatase (ALP) activities. BPA interrupts the bone metabolism via RANKL, apoptosis and Wnt/β-catenin signaling pathways. It is, however, still debated on the exact underlying mechanism of BPA against bone health. We summarised the molecular evidences with possible mechanisms of BPA, an old environmental culprit, in bone loss and enlightened the underlying understanding of adverse action of BPA in the society. © 2018 Elsevier Inc.

Objectives: The aim of the current study was to investigate the effects of berberine chloride and various heat conditions on the gene expression of Osterix, RUNX-2, RANKL, CDK2, CDK4, IL-6 and IL-11. Methods: Six groups of cells were treated with hyperthermia for 1 h: Two groups at mild, two at moderate and two at severe hyperthermia (39, 43 and 45°C respectively). Berberine chloride (80 µg/mL) was selected for treating one group of mild, moderate and severe hyperthermia (combination). All treated groups were recovered at 37°C for 24 h. Another exposure for hyperthermia (1 h) and recovery for 3 h at 37°C were applied. Results: The expression of Osterix was highly upregulated in all groups except in the severe hyperthermia and mild hyperthermia with berberine groups. Only the mild hyperthermia without berberine induced a slight increase in the expression of RUNX2, whereas severe hyperthermia alone suppressed the levels on a significant manner. Berberine alone was more effective in significantly up-regulating RANKL expression. On the other hand, CDK2 mRNA was downregulated in all groups. CDK4 showed a similar regulation in the mild hyperthermia group with control, but the expression was downregulated in the other groups, especially in severe hyperthermia the expression was significantly downregulated (p

The role of HDL in reverse cholesterol transport is well known but other atheroprotective mechanism, e.g. antioxidant capacities of HDL is not clear. The aim of this study is to determine the effects of different doses of HDL on antioxidant activities assay. HDL was isolated from plasma by single step-ultracentrifugation. The antioxidant activity of different doses of HDL were measured by ferric thiocyanate (FTC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and dichlorofluorescein (DCF) tests. Coincubation of HDL with LDL showed longer lag time and lower reaction rate in a dose-dependent manner compared to LDL alone (p

Inhibition of telomerase activity has emerged as a promising strategy to combat cancer cells, especially ones with no specific molecular targets such as triple negative breast cancer (TNBC). Cisplatin, a chemotherapeutic drug, is causing DNA damage while apigenin, a plant-derived antioxidant, induces apoptosis in various cancer cell types. Little is known about their combined ability to inhibit telomerase activity in TNBC cells. In the current study, the effect of cisplatin in combination with apigenin was investigated with regards to telomerase activity and expression of the telomerase catalytic subunit hTERT as well as Heat Shock Protein 90 (Hsp90) and p23 in two types of TNBC (MDA-MB-231; HCC1806) and one non-tumorigenic (MCF10A) epithelial cell line. The results showed that the combined treatment of cisplatin and apigenin significantly down-regulated telomerase activity. The inhibition of telomerase activity was accompanied by a down-regulation of hTERT, Hsp90 and p23 at transcriptional and translational level in both TNBC cells, as compared to control cells. The results of the current study suggest that apigenin and cisplatin synergistically inhibit telomerase activity by downregulating the enzyme’s catalytic subunit. However, the exact roles of Hsp90 and p23 in the regulation of telomerase activity requires further investigation as they seem to be TNBC subtype-specific.

This article reports the in vitro degradation and cytotoxicity assessment of Zn–3Mg alloy developed for biodegradable bone implants. The alloy was prepared using casting, and its microstructure was composed of Mg2Zn11 intermetallic phase distributed within a Zn-rich matrix. The degradation assessment was done using potentiodynamic polarization and electrochemical impedance spectrometry. The cell viability and the function of normal human osteoblast cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and alkaline phosphatase extracellular enzyme activity assays. The results showed that the degradation rate of the alloy was slower than those of pure Zn and pure Mg due to the formation of a high polarization resistance oxide film. The alloy was cytocompatible with the normal human osteoblast cells at low concentrations (

The recent proposal of using Zn-based alloys for biodegradable implants was not supported with sufficient toxicity data. This work, for the first time, presents a thorough cytotoxicity evaluation of Zn-3Mg alloy for biodegradable bone implants. Normal human osteoblast cells were exposed to the alloy's extract and three main cell-material interaction parameters: cell health, functionality and inflammatory response, were evaluated. Results showed that at the concentration of 0.75mg/ml alloy extract, cell viability was reduced by ~50% through an induction of apoptosis at day 1; however, cells were able to recover at days 3 and 7. Cytoskeletal changes were observed but without any significant DNA damage. The downregulation of alkaline phosphatase protein levels did not significantly affect the mineralization process of the cells. Significant differences of cyclooxygenase-2 and prostaglandin E2 inflammatory biomarkers were noticed, but not interleukin 1-beta, indicating that the cells underwent a healing process after exposure to the alloy. Detailed analysis on the cell-material interaction is further discussed in this paper.

In the present study, bioactive composites composed of hydroxyapatite (HA) and zeolite-Y were successfully synthesized by using a cost-effective microwave-assisted wet precipitation method. The phase purity, functional groups, surface topography, and elemental composition of the composite materials were examined by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), and energy dispersive X-ray spectroscopy (EDX), respectively. The XRD, FTIR, and EDX results confirmed the presence of zeolite with HA, demonstrating the formation of zeolite–HA composites. FESEM findings revealed that the particles had spherical morphology. The in vitro bioactivity assay using simulated body fluid (SBF) showed that the silica-based materials had the ability to support and accelerate the formation of dense layer. The cell viability of normal human osteoblast (NHOst) cells on the composites was confirmed by using cytotoxicity assay. In addition, FESEM results affirmed that the material supported adhesion of NHOst cells on its surface. In conclusion, the nano-structured zeolite–HA composites developed in this study showed good bioactivity and in vitro cell compatibility, and, therefore, may be considered as a potential candidate for bone tissue engineering applications.

This study investigates the effect of ACE2 activation on leptin-induced changes in systolic blood pressure (SBP), proteinuria, endothelial activation and ACE2 expression during pregnancy in Sprague-Dawley rats. Pregnant rats were given subcutaneous injection of either saline, or leptin, or leptin plus xanthenone (ACE2 activator), or xanthenone (XTN) alone. SBP, serum ACE, ACE2, endothelin-1, E-selectin and ICAM-1 levels were estimated; also their gene expressions were determined in the kidney and aorta respectively. Compared to control, SBP was higher in the leptin-only treated group (P

Anti-Vascular Endothelial Growth Factors (Anti-VEGF) agents have received recent interest as potential anti-fibrotic agents for their concurrent use with trabeculectomy. Preliminary cohort studies have revealed improved bleb morphology following trabeculectomy augmented with ranibizumab. The effects of this humanized monoclonal antibody on human Tenon's fibroblast (HTF), the key player of post trabeculectomy scar formation, are not fully understood. This study was conducted to understand the effects of ranibizumab on extracellular matrix production by HTF. The effect of ranibizumab on HTF proliferation and cell viability was determined using MTT assay (3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium). Ranibizumab at concentrations ranging from 0.01 to 0.5 mg/mL were administered for 24, 48 and 72 h in serum and serum free conditions. Supernatants and cell lysates from samples were assessed for collagen type 1 alpha 1 and fibronectin mRNA and protein level using quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). After 48-h, ranibizumab at 0.5 mg/mL, significantly induced cell death under serum-free culture conditions (p 0.05). Ranibizumab caused significant reduction of collagen type 1 alpha 1 (COL1A1) mRNA, but not for fibronectin (FN). Meanwhile, COL1A1 and FN protein levels were found upregulated in treated monolayers compared to control monolayers. Ranibizumab at 0.5 mg/mL significantly reduced cell viability in cultured HTF. From this study, we found that single application of ranibizumab is inadequate to induce the anti-fibrotic effects on HTF, suggesting the importance of adjunctive therapy. Further studies are underway to understand mechanism of actions of ranibizumab on HTF.

We investigated the possibility of enhancing hydroxyapatite (HA) bioactivity by co-substituting it with zinc and silver. Zn–Ag–HA nanoparticles were synthesized by using the microwave-assisted wet precipitation process, and their phase purity, elemental composition, morphology, and particle size were analyzed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX). FTIR, XRD, and EDX results showed the characteristic peaks of the Zn–Ag–HA structure, while SEM results demonstrated that the nanoparticles were of spherical shape with a particle size of 70–102 nm. Antibacterial tests of the nanoparticles revealed their antibacterial activity against Staphylococcus aureus and Escherichia coli. By using simulated body fluid (SBF), an apatite layer formation was observed at 28 days. In vitro cell adhesion assay confirmed the cell attachment of normal human osteoblast (NHOst) cells to the disc surface. MTT [(3(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide] assay indicated that the cells were viable, and the cells proliferated faster on the disks than on the control surface due to the presence of metal ions. In conclusion, the novel Zn–Ag–HA nanoparticles were found to be compatible with in vitro experiments and having potential antibacterial properties. Therefore these nanoparticles could be a promising candidate for future biomedical applications.

The structure and functions of endothelial cells after space mission were studied by electron and laser confocal microscopy, image analysis, and MTT test. The endothelial cells changed significantly (proliferative activity, size, contours, shape, distribution of mitochondria and microtubules) in comparison with controls on the Earth. These changes indicated injuries in the cytoskeleton and impairment of the barrier function of the cells, which presumably contributed to the development of endothelial dysfunction.

Background This study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM). Material/Methods Fifty female mice, aged 4–6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3. Results The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p

Background: Hypermethylation in promoter regions of genes might lead to altered gene functions and result in malignant cellular transformation. Thus, biomarker identification for hypermethylated genes would be very useful for early diagnosis, prognosis, and therapeutic treatment of oral squamous cell carcinoma (OSCC). The objectives of this study were to screen and validate differentially hypermethylated genes in OSCC and correlate the hypermethylation-induced genes with demographic, clinocopathological characteristics and survival rate of OSCC. Methods: DNA methylation profiling was utilized to screen the differentially hypermethylated genes in OSCC. Three selected differentially-hypermethylated genes of p16, DDAH2 and DUSP1 were further validated for methylation status and protein expression. The correlation between demographic, clinicopathological characteristics, and survival rate of OSCC patients with hypermethylation of p16, DDAH2 and DUSP1 genes were analysed in the study. Results: Methylation profiling demonstrated 33 promoter hypermethylated genes in OSCC. The differentially-hypermethylated genes of p16, DDAH2 and DUSP1 revealed positivity of 78%, 80% and 88% in methylation-specific polymerase chain reaction and 24% and 22% of immunoreactivity in DDAH2 and DUSP1 genes, respectively. Promoter hypermethylation of p16 gene was found significantly associated with tumour site of buccal, gum, tongue and lip (P=0.001). In addition, DDAH2 methylation level was correlated significantly with patients' age (P=0.050). In this study, overall five-year survival rate was 38.1% for OSCC patients and was influenced by sex difference. Conclusions: The study has identified 33 promoter hypermethylated genes that were significantly silenced in OSCC, which might be involved in an important mechanism in oral carcinogenesis. Our approaches revealed signature candidates of differentially hypermethylated genes of DDAH2 and DUSP1 which can be further developed as potential biomarkers for OSCC as diagnostic, prognostic and therapeutic targets in the future.

Background Oral cancer has become one of the most prevalent cancers worldwide and human Papillomavirus is one of the risk factors for developing oral cancer. For this study HPV18 was chosen as it is one of the high risk HPV types and may lead to carcinogenesis. However, prevalence of HPV18 infection in Oral Squamous Cell Carcinoma in Malaysia remains unclear. Objective This study aimed to investigate the viral load of HPV18 DNA in OSCC and potentially malignant lesions using saliva samples. Materials and methods Genomic DNAs of thirty saliva samples of normal subjects and thirty saliva samples compromised of 16 samples from potentially malignant lesions and 14 of OSCC patients were amplified for HPV18 DNA using a nested polymerase chain reaction analysis. All PCR products were then analyzed using the Bioanalyzer to confirm presence of HPV18 DNA. Result From thirty patients examined, only one of 30 (3.3%) cases was found to be positive for HPV18 in this study. Conclusion The finding of this study revealed that there is a low viral detection of HPV18 in Malaysian OSCC by using saliva samples, suggesting that prevalence of HPV18 may not be important in this group of Malaysian OSCC.

This study was conducted to investigate the effects of high-density lipoprotein (HDL) onsecretion and gene expression of intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial-leukocyte adhesion molecule-1 (E-selectin), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) by human vascular endothelial cells (HUVEC). Lipoploysacharides-stimulated HUVEC was incubated with different concentrations of HDL (20-120 mg/dL) for 16 hours. The levels of all biomarkers were measured by ELISA. Total RNA was extracted and all bioamarkers’ mRNA levels were measured by Taqman real-time polymerase chain reaction assay. Different HDL concentrations reduced the secretion and expressonof all biomarkers except VCAM-1, which may contribute to the prevention and regression of atherosclerosis.Keywords: HDL; adhesion molecules; cytokines; endothelial cells; lipopolysaccharides.

This study investigates the effect of adiponectin on endothelial activation and inflammatory marker secretion by human coronary artery endothelial cells (HCEAC) in vitro. Methodology : HCAEC at the seventh passage were divided into two groups and incubated or 24 hours at 37° C and 5% CO2 as follows: Control, and adiponectin-treated (30 μg/ml adiponectin) groups. Supernatants were analysed for ICAM-1, E-selectin, PAI-1 and IL-6 and COX-2 using ELISA. RT-PCR was used to analyse gene expression of ICAM-1, E-selectin, PAI-, IL-6, COX-2, NFkBp50 and NFkBp65. Data were analysed using independent t-test. Results : ICAM-1 and E-selectin level was significantly higher in leptin-adiponectin-treated groups (P

Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases.

Osteoporosis is a chronic and progressive disease of multifactorial etiology and is the most common metabolic bone disease in the United States and the world at large. Palm vitamin E has been repor...

Introduction: Cyclophosphamide (CPA) chemotherapy leads to ovarian failure and infertility. Tocotrienol (T3) is an antioxidant and anti-inflammatory agent. The role of T3 in ovarian protection throughout chemotherapy remains unclear. Aim: To investigate the role of T3 in the preservation of female fertility in CPA treatment. Method: Sixty female mice were divided into five treatment groups, namely, normal saline, corn oil only, T3 only, CPA and CPA + T3. The treatment was given for 30 days, followed by administration of gonadotrophin to induce ovulation. After killing, both ovaries were collected and examined histologically. Results: There was significant reduction in ovarian size in the CPA group compared with the normal group (CPA versus normal, mean area ± SD; 0.118 ± 0.018 vs. 0.423 ± 0.024 cm2; p ≤ 0.005), whilst concurrent administration of T3 with CPA leads to conservation of ovarian size (CPA + T3 vs. CPA, mean area ± SD; 0.285 ± 0.032 vs. 0.118 ± 0.018 cm2; p ≤ 0.005). Ovaries in CPA group showed abnormal folliculogenesis with accompanied reduced ovulation rate, follicular oedema, increased vascularity and inflammatory cell infiltration. These changes were reversed by concurrent T3 administration. Conclusion: Co-administration of T3 with CPA confers protection of ovarian morphology and function in vivo. These findings contribute to the further elucidation of CPA effect on ovary and suggest the potential of T3 use in preserving fertility in chemotherapy.

Coscinium fenestratum is a woody climber found in south East Asia and has been widely used as a medicinal plant. The infusion and tincture preparation of stem is widely used in traditional medicine. Our research aims to investigate the effect of Coscinium fenestratum ( C. fenestratum ) stem extract on plasma glucose, biochemical parameters, antioxidant enzymes and proteome in streptozotocin (STZ) induced diabetic rats. Crude extract from C. fenestratum stem was extracted using Dichloromethane (DCM) and fractionated into fractions using column chromatography. The glucose lowering effect was examined using STZ-induced rats and biochemical parameters and antioxidant enzymes assessed using commercial laboratory services. Proteome analysis was done using two dimensional gel electrophoresis (2-DE) and mass spectrometry (Proteomics technique). Protein–protein interaction network (PPI) was determined by multidimensional protein identification technology (MudPIT) and the interaction between the proteins were analyzed using STRING v10 database. Our findings showed that C. fenestratum stem extract was able to reduce the plasma glucose level, maintain the kidney and liver biomarkers and increase the antioxidant enzymes. Proteome analysis showed elevation of lipid metabolizing proteins, channel proteins, protection proteins, antioxidant proteins, and digestive proteins in the treated group. HPLC analyses of purified fraction E from the stem extract of C. fenestratum have shown that Palmatine is one of the major bioactive components. C. fenestratum stem extract is able to reduce the plasma glucose level, reduce lipids, increase antioxidant enzymes and stimulated the very important lipid metabolizing proteins, channel proteins, protection proteins, antioxidant proteins, and digestive proteins.

Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35 °C) and severe (27 °C) hypothermia for 1, 12, 24 and 72 h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p

After chronic, low-frequency stimulation, a rapid decline in Ca2+pump activity is observed during the early stages of skeletal muscle transformation. However, this variation in enzymatic activity does not coincide with a drastic reduction in the amount of sarcoplasmic reticulum Ca2+-ATPases. To investigate whether changes in subunit interactions within Ca2+pump complexes contribute to this phenomena, we performed a chemical cross-linking analysis of 4 days continuously, and 4 days discontinuously, electrostimulated fast muscle fibers. The abundance of the slow and fast Ca2+-ATPase isoforms sarco(endo)plasmic reticulum Ca2+- ATPase types 1 and 2 was affected during the fast-to-slow transition process, demonstrating that, even after short-term stimulation, distinct changes in the isoform expression pattern of muscle proteins occur. However, the oligomeric status of both ion pump species did not change. Hence, chemical modifications of critical enzyme domains must be responsible for the rapid stimulation-induced activity changes, not variations in protein-protein interactions within Ca2+-ATPase units. Oligomerization appears to be of central importance to the proper physiological functioning of the Ca2+-ATPase and does not undergo changes during skeletal muscle conditioning.

Using a biochemical/immunological approach to analyse the protein constituents of skeletal-muscle junctional-face membrane (JFM), we identified a 45kDa protein. Its N-terminal amino acid was blocked, but the amino acid sequence obtained from several peptides after proteolytic treatment did not significantly match that of any protein present in the SwissProt and NCBI (National Center for Biotechnology Information) databases. We synthesized a peptide whose sequence matched that of one of the peptides obtained after CNBr cleavage of the 45kDa protein; the peptide was conjugated to a carrier and used to raise antibodies. The antiserum was used to study in more detail the biochemical characteristics of the novel 45kDa protein. Analysis of the proteins present in different subcellular membrane fractions show that the novel 45kDa polypeptide: (i) is an integral membrane constituent present both in neonatal and adult skeletal-muscle sarcoplasmic reticulum; (ii) is selectively localized in the JFM; (iii) is not present in microsomes obtained from rabbit heart, liver or kidney. Immunoprecitation with anti-(45kDa protein) antibody indicates that the 45kDa protein is part of a complex which can be phosphorylated in vitro by the catalytic subunit of protein kinase A.

Although all muscle cells generate contractile forces by means of organized filament systems, isoform expression patterns of contractile and regulatory proteins in heart are not identical compared to developing, conditioned or mature skeletal muscles. In order to determine biochemical parameters that may reflect functional variations in the Ca(2+)-regulatory membrane systems of different muscle types, we performed a comparative immunoblot analysis of key membrane proteins involved in ion homeostasis. Cardiac isoforms of the alpha(1)-dihydropyridine receptor, Ca(2+)-ATPase and calsequestrin are also present in skeletal muscle and are up-regulated in chronic low-frequency stimulated fast muscle. In contrast, the cardiac RyR2 isoform of the Ca(2+)-release channel was not found in slow muscle but was detectable in neonatal skeletal muscle. Up-regulation of RyR2 in conditioned muscle was probably due to degeneration-regeneration processes. Fiber type-specific differences were also detected in the abundance of auxiliary subunits of the dihydropyridine receptor, the ryanodine receptor and the Ca(2+)-ATPase, as well as triad markers and various Ca(2+)-binding and ion-regulatory proteins. Hence, the variation in innervation of different types of muscle appears to have a profound influence on the levels and pattern of isoform expression of Ca(2+)-regulatory membrane proteins reflecting differences in the regulation of Ca(2+)-homeostasis. However, independent of the muscle cell type, key Ca(2+)-regulatory proteins exist as oligomeric complexes under native conditions.

The skeletal muscle excitation-contraction-relaxation cycle matures during the first weeks after birth and protein-protein interactions are believed to be essential for proper Ca2+ regulation. We therefore studied potential changes in the oligomerisation of key components of the Ca2+-regulatory membrane system during postnatal myogenesis. In contrast to a decrease in calreticulin, the Ca2+-binding proteins calsequestrin and sarcalumenin increased in abundance in microsomes isolated from muscle between postnatal days 1 and 41. While the expression of the fast Ca2+-ATPase increased, its slow-twitch isoform decreased. The junctional component triadin, the 53 kDa sarcoplasmic reticulum glycoprotein, as well as the dihydropyridine receptor increased in abundance, while no major changes in the expression of the ryanodine receptor were observed. Crosslinking analysis revealed that the fast Ca2+-ATPase, alpha1-dihydropyridine receptor and calsequestrin exhibit a more pronounced tendency to oligomerise in adult muscle fibres as compared to early postnatal stages. Interestingly, adult calsequestrin exists not only as a 63 kDa form but also as stable molecular species of higher molecular mass. These findings imply that during postnatal development, protein-protein interactions within the Ca2+-regulatory membrane system become more complex and oligomerisation appears to be an essential prerequisite for the proper physiological functioning of key membrane proteins in matured skeletal muscle fibres.

The ability of phytochemicals to suppress production of superoxide anion (O 2 − ) in a cell culture model was assessed. Differentiated U937 cells and human polymorph nuclear leukocytes (PMN) were stimulated with increasing concentrations of phorbol myristate acetate (PMA) and O 2 − release was measured by cytochrome c reduction. The overall trend in O 2 − production in response to PMA stimulation was similar in both U937 cells and PMN. The effect of the phytochemicals, chlorogenic acid, caffeic acid, curcumin, quercetin, (-)-epicatechin and glycyrrhizic acid (50, 100 and 500 nM) on O 2 − formation from PMA stimulated U937 cells was examined. All phytochemicals suppressed O 2 − release from U937 cells with chlorogenic acid, (-)-epicatechin and glycyrrhizic acid showing dose dependent effects. In conclusion, U937 cells are a useful model to screen phytochemicals for their ability to suppress O 2 − formation. © 1997 Elsevier Science Inc.