This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.
Cartilage is regularly needed for reconstructive surgery. Basic research in tissue engineering is necessary to develop its full potential. We presented here the expression profile of type II collagen gene and type I collagen gene in human auricular monolayer culture expansion. Cultured chondrocytes documented a reduction in the expression level of collagen type II gene whilst collagen type I gene was gradually expressed through all the passages. This study demonstrated that human auricular chondrocytes lose its phenotypic expression during monolayer culture expansion. Further studies are required to enhance cartilage specific gene expression, collagen type II throughout the in vitro culture.
To date there is no optimal approach to reconstruct an external ear. However, advances in tissue engineering technologies have indicated that in vitro autologous elastic cartilage might be of great importance in the future treatment of these patients. The aim of this study was to observe monolayer expansion of auricular cartilage and to evaluate engineered cartilage using standard histochemical study.
Virus isolation and accurate characterization plays a crucial role in the rapid identification of the causative agents of infectious disease outbreaks especially if the causative viruses are novel where no pre-existing diagnostic reagents would be available. A new cell culture tube, named Jui Meng (JM) Cell Culture Tube, was developed to reduce the cost and improve the efficiency and biosafety of work pertaining to virus isolation. The design of the tube is based heavily on the principle of practicability, functionality, biosafety and long-term cost saving for diagnostic laboratory work in virus isolation. It is designed to culture an initial inoculum of one milliliter of culture medium containing 1 x 10(4) to 1 x 10(5) cells/ml.
The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H2O2) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l(-1), intracellular AQ content at 42 mg g(-1) DW, and H2O2 level at 9 micromol g(-1) FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g(-1) FW and total carotenoids at 13.3 mg g(-1) FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H2O2 levels. Vitamin C content at 30-120 microg g(-1) FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400-500 microg g(-1) FW in 7-day-old cultures from all medium strategies and reduced to 50-150 microg g(-1) FW on day 14 and 21; as compared to 60 microg g(-1) FW in callus and 200 microg g(-1) FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.
The enhancement of cell proliferation and promotion of cell survival via the inhibition of apoptosis is thought to be the key to the initiation and progression of cancers. The phosphatidylinositol-3 kinase (PI3K)/Akt is an important survival signal pathway that has been shown to be crucial in the regulation of balance between pro-apoptotic and survival (anti-apoptotic) signal. In this study, the expression of phosphorylated Akt at Thr308 and Ser473, BCL-2-antagonist of cell death (BAD) at Ser136 and glycogen synthase kinase-3beta (GSK-3beta) at Ser9 in 47 paraffin-embedded human colorectal carcinoma (CRC) tissues were determined by immunohistochemical staining in order to dissect the alterations in the signal transduction pathways in CRC. Our results showed that there was a significant increase in the expression of these biomolecules in CRC tissues compared to the apparently normal adjacent tissues. The frequency of increased expression in tumor colonic mucosa were as follows: p-Akt1/2/3 (Thr308) = 16/47 (34%); p-Akt1 (Ser473) = 21/47 (44.7%); phospho-BAD (p-BAD) Ser136 = 27/47 (57.4%) and phospho-GSK-3beta (p-GSK-3beta) = 21/47 (44.7%). Analysis of the total p-Akt1 (Ser473), p-Akt1/2/3 (Thr308), p-GSK-3beta (Ser9) and p-BAD (Ser136) score found that there was a statistically significant relationship with each other. A statistically significant positive linear relationship was found between total p-Akt (Ser473) score and total p-GSK-3beta (Ser9) score as well as with total p-BAD (Ser136) score. On the other hand, total p-Akt1/2/3 (Thr308) scores had a statistically significant positive linear relationship with p-GSK-3beta (Ser9) only. The Akt targets, p-GSK-3beta (Ser9) and p-BAD (Ser136) were positively correlated to each other. There was no significant correlation between clinico-pathological data with total p-Akt1 (Ser473), p-Akt1/2/3 (Thr308), p-GSK-3beta (Ser9) and p-BAD (Ser136) score except for age. The total scores of p-GSK-3beta were found to be higher in patients in the age group of greater than 60. This is the first report of p-Akt1/2/3 (Thr308) and p-BAD (Ser136) expression in primary colorectal tumor tissue. Our data further supports the role of PI3K/Akt signaling pathways in the pathogenesis of CRC and contributes to the identification of target molecules in the signal transduction pathway for cancer therapy.
Vitamin E is important not only for its cellular antioxidant and lipid-lowering properties, but also as an antiproliferating agent. It has also been shown to contribute to immunoregulation, antibody production, and resistance to implanted tumors. It has recently been shown that tocotrienols are the components of vitamin E responsible for growth inhibition in human breast cancer cells in vitro as well as in vivo through estrogen-independent mechanisms. Although tocotrienols act on cell proliferation in a dose-dependent manner and can induce programmed cell death, no specific gene regulation has yet been identified. In order to investigate the molecular basis of the effect of a tocotrienol-rich fraction (TRF) from palm oil, we performed a cDNA array analysis of cancer-related gene expression in estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cells. The human breast cancer cells were incubated with or without 8 mug/mL of tocotrienols for 72 h. RNA was subsequently extracted and subjected to reverse transcription before being hybridized onto cancer arrays. Tocotrienol supplementation modulated significantly 46 out of 1200 genes in MDA-MB-231 cells. In MCF-7 cells, tocotrienol administration was associated with a lower number of affected genes. Interestingly, only three were affected in a similar fashion in both cell lines: c-myc binding protein MM-1, 23-kDa highly basic protein, and interferon-inducible protein 9-27 (IFITM-1). These proteins are most likely involved in the cell cycle and can exert inhibitory effects on cell growth and differentiation of the tumor cell lines. These data suggest that tocotrienols are able to affect cell homeostasis, possibly independent of their antioxidant activity.
The oxidative response of Burkholderia pseudomallei and Escherichia coli infected macrophages from normal and melioidosis subjects was determined by measuring the production of nitric oxide which is one of the reactive nitrogen intermediates, and the activation state of these macrophages was determined by measuring the generation of 8-iso-PGF(2alpha), a bioactive product of free radical induced lipid peroxidation. Macrophages obtained from the melioidosis patients generated significantly lower levels of nitric oxide and 8-iso-PGF(2alpha) compared to macrophages obtained from the normal subjects (P<0.001). The reduced efficiency of the oxygen dependent microbicidal mechanism in macrophages of melioidosis patients may be one of the survival strategies developed by B. pseudomallei to remain viable intracellularly.
Three new fully dehydrogenated naphthylisoquinoline alkaloids, the 7,1'-coupled ent-dioncophylleine A (3a), the likewise 7,1'-coupled 5'-O-demethyl-ent-dioncophylleine A (4), and the 7,8'-linked dioncophylleine D (5), have been isolated from the leaves of the recently described Malaysian highland liana Ancistrocladusbenomensis. All of them lack an oxygen function at C-6; this so-called Dioncophyllaceae-type structural subclass had previously been found only in naphthylisoquinoline alkaloids from West and Central African plants. Moreover, compounds 3a and 4 are the first fully dehydrogenated, i.e., only axially chiral, naphthylisoquinoline alkaloids of this type that are optically active; compound 5, by contrast, is fully racemic, due to its configurationally unstable biaryl axis. The structural elucidation was achieved by spectroscopic and chiroptical methods. Biological activities of these alkaloids against different protozoan parasites are described.
This study was to investigate the effects of insulin-transferrin-selenium (ITS) on the proliferation and quantitative gene expression of adult human nasal septum chondrocytes in monolayer culture expansion and the formation of tissue engineered hyaline cartilage. Effects of ITS on human nasal septum chondrocytes monolayer culture expansion and gene expression were evaluated in various culture media either added with 2% fetal bovine serum (FBS) or 1 ng/mL basic fibroblast growth factor plus 1 ng/mL transforming growth factor or both serum and growth factors supplementation in comparison with medium added with 10%FBS. Chondrocytes cultured in medium added with 2% fetal bovine serum and growth factors either supplemented with or without ITS were then mixed with pluronic F-127 hydrogel for in vivo tissue engineered cartilage formation in nude mice model. Engineered tissues were removed after 8 weeks of implantation and evaluated with histological staining, immunohistochemistry, transmission electron microscopy and quantitative gene expression analysis. ITS promoted human chondrocytes proliferation and reduced chondrocytes dedifferentiation in media supplemented with serum and growth factors. ITS with 2% FBS and growth factors provided 15-fold increased in chondrocytes number by the end of the culture period compared to the standard culture medium used in chondrocytes culture (medium added with 10% FBS). Engineered tissue resulted from ITS supplementation demonstrated higher quality of cartilage formation. In conclusion, our study has demonstrated the benefits of ITS supplementation in human chondrocytes monolayer culture and tissue engineering cartilage formation.
The nucleocapsid (N) protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use as a diagnostic reagent, the NiV N gene was cloned into the pFastBacHT vector and the His-tagged fusion protein was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-NiV antibodies produced a band of approximately 62 kDa. A time course study showed that the highest level of expression was achieved after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel-nitrilotriacetic acid resin column revealed different types of structures, including spherical, ring-like, and herringbone-like particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hyrdrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein revealed a high A(260)/A(280) ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay results showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition.
Treatment and management of congenital as well as post-traumatic trachea stenosis remains a challenge in pediatric surgery. The aim of this study was to reconstruct a trachea with human nasal septum chondrocytes by using the combination of biodegradable hydrogel and non-biodegradable high-density polyethylene (HDP) as the internal predetermined shape scaffold.
In the present study we examined the effect of E. longifolia methanol extract (TA164) on the GSH levels of P. falciparum infected erythrocytes and uninfected erythrocytes. Our study on parasite growth shows the IC50 and IC75 values of TA164 to be 0.17 g/ml and 6 g/ml respectively while for BSO was 25.5 g/ml and 46.5 g/ml respectively. About 95% to 100% growth inhibition of P. falciparum infected erythrocyte was observed when treated with TA164 and BSO at 16 g/ml and 64 g/ml respectively. The study on GSH contents indicated that non-infected erythrocytes treated with 6 g/ml (IC75 values) of TA164 at 24 hours incubation showed less GSH content as compared to non-treated erythrocytes. A similar observation was seen on treated trophozoite infected erythrocyte (10% parasitemia) when treated with 6 g/ml at 3 hours incubation. Analysis of the GSH contents of parasite compartments treated with TA164 at the same concentration (6 g/ml) for 3 hours incubation indicated a reduction of GSH contents. At the same concentration, TA164 did not affect the GSH contents of enriched trophozoite infected erythrocytes (60-70% parasitemia). TA164 did affect the GSH content of non-infected erythrocyte at 24 hours (accept IC50 value) as well as the parasite compartments (trophozoite infected erythrocyte and parasite itself) but fails to affect the GSH content of enriched trophozoite infected erythrocyte.
The effects of tocotrienols on murine liver cell viability and their apoptotic events were studied over a dose range of 0-32 microg mL(-1). Normal murine liver cells (BNL CL.2) and murine liver cancer cells (BNL 1ME A.7R.1) were treated with tocotrienols (T(3)), alpha tocopherol (alpha-T) and the chemo drug, Doxorubicin (Doxo, as a positive control). Cell viability assay showed that T(3) significantly (P < or = 0.05) lowered the percentage of BNL 1ME A.7R.1 cell viability in a dose-responsive manner (8-16 microg mL(-1)), whereas T did not show any significant (P>0.05) inhibition in cell viability with increasing treatment doses of 0-16 microg mL(-1). The IC(50) for tocotrienols were 9.8, 8.9, 8.1, 9.7, 8.1 and 9.3 microg mL(-1) at 12, 24, 36, 48, 60 and 72 hours respectively. Early apoptosis was detected 6 hours following T(3) treatment of BNL 1ME A.7R.1 liver cancer cells, using Annexin V-FITC fluorescence microscopy assay for apoptosis, but none were observed for the non-treated liver cancer cells at the average IC(50) of 8.98 microg mL(-1) tocotrienols for liver cancer cells. Several apoptotic bodies were detected in BNL 1ME A.7R.1 liver cancer cells at 6 hours post-treatment with tocotrienols (8.98 microg mL(-1)) using Acridine Orange/Propidium Iodide fluorescence assay. However, only a couple of apoptotic bodies were seen in the non-treated liver cancer cells and the BNL CL.2 normal liver cells. Some mitotic bodies were also observed in the T(3)-treated BNL 1ME A.7R.1 liver cancer cells but were not seen in the untreated BNL 1ME A.7R.1 cells and the BNL CL.2 liver cells. Following T(3)-treatment (8.98 microg mL(-1)) of the BNL 1ME A.7R.1 liver cancer cells, 24.62%, 25.53% and 44.90% of the cells showed elevated active caspase 3 activity at 9, 12 and 24 hours treatment period, respectively. DNA laddering studies indicated DNA fragmentation occurred in the T(3)-treated liver cancer cells, BNL 1ME A.7R.1 but not in non-treated liver cancer cells and the T(3)-treated and non-treated normal liver cells. These results suggest that tocotrienols were able to reduce the cell viability in the murine liver cancer cells at a dose of 8-32 microg mL(-1) and that this decrease in percentage cell viability may be due to apoptosis.
The effect of infection with teratogenic viruses at early stages of pregnancy is not fully understood. This study aimed to look at the effect of infection with teratogenic viruses such as bovine viral diarrhea virus (BVDV) and border disease virus (BDV), on early stage embryos at the hatched blastocyst stage. BVDV and BDV are known to cross the placenta of infected mothers and lead to congenital defects and death of developing fetuses. This study can be a good model for better understanding the effects of other teratogenic viruses such as Rubella virus in humans.
Restenosis represents a major impediment to the success of coronary angioplasty. Abnormal proliferation of vascular smooth muscle cells (VSMCs) has been shown to be an important process in the pathogenesis of restenosis. A number of agents, particularly rapamycin and paclitaxel, have been shown to impact on this process. This study was carried out to determine the mechanisms of cytotoxicity of goniothalamin (GN) on VSMCs. Results from MTT cytotoxicity assay showed that the IC(50) for GN was 4.4 microg/ml (22 microM), which was lower compared to the clinically used rapamycin (IC(50) of 25 microg/ml [27.346 microM]). This was achieved primarily via apoptosis where up to 25.83 +/- 0.44% of apoptotic cells were detected after 72 h treatment with GN. In addition, GN demonstrated similar effects as rapamycin in inhibiting VSMCs proliferation using bromodeoxyuridine (BrdU) cell proliferation assay after 72 h treatment at IC(50) concentration (p > 0.05). In order to understand the mechanisms of GN, DNA damage detection using comet assay was determined at 2h post-treatment with GN. Our results showed that there was a concentration-dependent increase in DNA damage in VSMCs prior to cytotoxicity. Moreover, GN effects were comparable to rapamycin. In conclusion, our data show that GN initially induces DNA damage which subsequently leads to cytotoxicity primarily via apoptosis in VSMCs.
A cucumber green mosaic mottle virus (CGMMV) full-length clone was developed for the expression of Hepatitis B surface antigen (HBsAg). The expression of the surface displayed HBsAg by the chimeric virus was confirmed through a double antibody sandwich ELISA. Assessment of the coat protein composition of the chimeric virus particles by SDS-PAGE analysis showed that 50% of the coat proteins were fused to the HBsAg. Biological activity of the expressed HBsAg was assessed through the stimulation of in vitro antibody production by cultured peripheral blood mononuclear cells (PBMC). PBMC that were cultured in the presence of the chimeric virus showed up to an approximately three-fold increase in the level of anti HBsAg immunoglobulin thus suggesting the possible use of this new chimeric virus as an effective Hepatitis B vaccine.
Oxidative stress is thought to be one of the factors that cause neurodegeneration and that this can be inhibited by antioxidants. Since astrocytes support the survival of central nervous system (CNS) neurons, we compared the effect of alpha-tocopherol and gamma-tocotrienol in minimizing the cytotoxic damage induced by H(2)O(2), a pro-oxidant. Primary astrocyte cultures were pretreated with either alpha-tocopherol or gamma-tocotrienol for 1 h before incubation with 100 microM H(2)O(2) for 24 h. Cell viability was then assessed using the MTS assay while apoptosis was determined using a commercial ELISA kit as well as by fluorescent staining of live and apoptotic cells. The uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes were also determined using HPLC. Results showed that gamma-tocotrienol is toxic at concentrations >200 microM but protects against H(2)O(2) induced cell loss and apoptosis in a dose dependent manner up to 100 microM. alpha-Tocopherol was not cytotoxic in the concentration range tested (up to 750 microM), reduced apoptosis to the same degree as that of gamma-tocotrienol but was less effective in maintaining the viable cell number. Since the uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes is similar, this may reflect the roles of these 2 vitamin E subfamilies in inhibiting apoptosis and stimulating proliferation in astrocytes.
Many plant-derived natural compounds have been reported previously to inhibit the production of important pro-inflammatory mediators such as nitric oxide, prostaglandin E2, TNF-alpha and reactive oxygen species by suppressing inducible enzyme expression via inhibition of the mitogen-activated protein kinase pathway and nuclear translocation of critical transcription factors. This study evaluates the effects of atrovirinone [2-(1-methoxycarbonyl-4,6-dihydroxyphenoxy)-3-methoxy-5,6-di-(3-methyl-2-butenyl)-1,4-benzoquinone)], a benzoquinone that we have previously isolated from Garcinia atroviridis, on two cellular systems that are repeatedly used in the analysis of anti-inflammatory bioactive compounds, namely, RAW 264.7 macrophage cells and whole blood. Atrovirinone inhibited the production of both nitric oxide and prostaglandin E2 from LPS-induced and IFN-gamma-induced RAW 264.7 cells and whole blood, with inhibitory concentration (IC)50 values of 4.62 +/- 0.65 and 9.33 +/- 1.47 micromol/L, respectively. Analysis of thromboxane B2 (TXB2) secretion from whole blood stimulated by either the cyclooxygenase (COX)-1 or the COX-2 pathway showed that atrovirinone inhibits the generation of TXB2 by both pathways, with IC50 values of 7.41 +/- 0.92 and 2.10 +/- 0.48 micromol/L, respectively. Analysis of IC50 ratios showed that atrovirinone was more COX-2 selective in its inhibition of TXB2, with a ratio of 0.32. Atrovirinone also inhibited the generation of intracellular reactive oxygen species and the secretion of TNF-alpha from RAW 264.7 cells in a dose-responsive manner, with IC50 values of 5.99 +/- 0.62 and 11.56 +/- 0.04 micromol/L, respectively. Lipoxygenase activity was also moderately inhibited by atrovirinone. Our results suggest that atrovirinone acts on important pro-inflammatory mediators possibly by the inhibition of the nuclear factor-kappaB pathway and also by the inhibition of the COX/lipoxygenase enzyme activity.
Fluoxetine (FLX), a P-glycoprotein inhibitor with antimalarial activity, is promising candidate for reversing chloroquine/mefloquine (CQ/MQ) resistance. The Dd2 strain of CQ- and MQ-resistant Plasmodium falciparum was synchronized and assayed with various concentrations of CQ/MQ individually and in combination with FLX or verapamil (VPL). Our results indicated the 50% inhibitory concentration values of CQ and MQ were greatly lowered when FLX was used simultaneously. Isobolograms indicated that CQ-FLX combinations are more synergistic (mean fractional inhibitory concentration [FIC] index 0.55) than MQ-FLX (mean FIC index 0.64), and their synergistic effect was better than or at par with CQ-VPL (mean FIC index 0.88) and MQ-VPL (mean FIC index 0.60) combinations. We conclude that the FLX potentiates the CQ and MQ response on multidrug-resistant P. falciparum at clinically achievable concentrations.