Sahidan Senafi

Intrinsic anticarcinogenic effects of Piper sarmentosum ethanolic extract on a human hepatoma cell line

BackgroundPiper sarmentosum, locally known as kaduk is belonging to the family of Piperaceae. It is our interest to evaluate their effect on human hepatoma cell line (HepG2) for the potential of anticarcinogenic activity.ResultsThe anticarcinogenic activity of an ethanolic extract from Piper sarmentosum in HepG2 and non-malignant Changs liver cell lines has been previously determined using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (MTT) assays, where the IC50 value was used as a parameter for cytotoxicity. The ethanolic extract that showed anticarcinogenic properties in HepG2 cells had an IC50 of 12.5 μg mL-1, while IC50 values in the non-malignant Changs liver cell line were greater than 30 μg mL-1. Apoptotic morphological changes in HepG2 cells were observed using an inverted microscope and showed chromatin condensation, cell shrinkage and apoptotic bodies following May-Grunwald-Giemsas staining. The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 μg mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells). Following acridine orange and ethidium bromide staining, treatment with 10, 12 and 14 μg mL-1 of ethanolic extracts caused typical apoptotic morphological changes in HepG2 cells. Molecular analysis of DNA fragmentation was used to examine intrinsic apoptosis induced by the ethanolic extracts. These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells. However, the non-malignant Changs liver cell line produced no DNA fragmentation. In addition, the DNA genome was similarly intact for both the untreated non-malignant Changs liver and HepG2 cell lines.ConclusionTherefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis pathway in HepG2 cells in vitro.

Determination of the differentiation capacities of murines' primary mononucleated cells and MC3T3-E1 cells

BackgroundThe main morphological features of primitive cells, such as stem and progenitor cells, are that these cells consists only one nucleus. The main purpose of this study was to determine the differentiation capacities of stem and progenitor cells. This study was performed using mononucleated cells originated from murine peripheral blood and MC3T3-E1 cells. Three approaches were used to determine their differentiation capacities: 1) Biochemical assays, 2) Gene expression analysis, and 3) Morphological observations.ResultsWe found that both cells were able to differentiate into mature osteoblasts, as assayed by ALP activity. RT-PCR analysis showed the activation of the Opn gene after osteoblast differentiation. Morphological observations of both cells revealed the formation of black or dark-brown nodules after von Kossa staining. Nevertheless, only mononucleated cells showed the significant increase in TRAP activity characteristic of mature osteoclasts. The osteoclast-specific CatK gene was only upregulated in mononucleated cells. Morphological observations indicated the existence of multinucleated osteoclasts. Sca-1 was activated only in undifferentiated mononucleated cells, indicating that the cells were hematopoietic stem cells. In both cell lines, the housekeeping Gapdh gene was activated before and after differentiation.ConclusionThe isolated mononucleated cells were able to differentiate into both osteoblasts and osteoclasts; indicating that they are stem cells. On the other hand, MC3T3-E1 cells can only differentiate into osteoblasts; a characteristic of progenitor cells.

Characterization of Mononucleated Human Peripheral Blood Cells

Unspecialized cells that can renew themselves and give rise to multiple differentiated cell types are termed stem cells. The objective of this study was to characterize and investigate, through molecular and biochemical analyses, the stemness of cells derived from isolated mononucleated cells that originated from peripheral blood. The isolated mononucleated cells were separated according to their physical characteristics (adherent and suspension), after 4 to 7 days into a 14-day culturing period in complete medium. Our results revealed that adherent and suspension cells were positive for mesenchymal stem cell (MSC) and hematopoietic stem cell (HSC) markers, respectively. Differentiation of adherent cells into osteoblasts was associated with expression of the OPN gene and increasing ALP enzyme activity, while differentiation of suspension cells into osteoclasts was associated with expression of the TRAP gene and increasing TRAP enzyme activity. In conclusion, molecular and biochemical analyses showed that mononucleated cells consist of MSC (adherent) and HSC (suspension), and both cell types are able to differentiate into specialized cells from their respective lineage: osteoblast (MSC) and osteoclast (HSC).

In vitro chondrogenesis transformation study of mouse dental pulp stem cells

A major challenge in the application of mesenchymal stem cells in cartilage reconstruction is that whether the cells are able to differentiate into fully mature chondrocytes before grafting. The aim of this study was to isolate mouse dental pulp stem cells (DPSC) and differentiate them into chondrocytes. For this investigation, morphological, molecular, and biochemical analyses for differentiated cells were used. To induce the chondrocyte differentiation, DPSC were cultured in chondrogenic medium (Zen-Bio, Inc.). Based on morphological analyses using toluidine blue staining, proteoglycan products appear in DPSC after 21 days of chondrocyte induction. Biochemical analyses in differentiated group showed that alkaline phosphatase activity was significantly increased at day 14 as compared to control (P < 0.05). Cell viability analyses during the differentiation to chondrocytes also showed that these cells were viable during differentiation. However, after the 14th day of differentiation, there was a significant decrease (P < 0.05) in the viability proportion among differentiated cells as compared to the control cells. In RT-PCR molecular analyses, mouse DPSC expressed Cd146 and Cd166 which indicated that these cells belong to mesenchymal stem cells. Coll I and Coll II markers showed high expression after 14 and 21 days, respectively. In conclusion, this study showed that DPSC successfully differentiated into chondrocytes.

A nationwide biotechnology outreach and awareness program for Malaysian high schools

Biotechnology education in developing nations remains one of the rate limiting factors in achieving optimal human resource capacity to drive and tap the bio-resources of these nations. Many developing countries are situated within rich bio-diversity enclaves. Biotechnology offers the promise of tapping these bio resources towards due process of developing these nations. While there may be a steady stream of biology and biotechnology based graduates, from Malaysian as well as foreign universities contributing to the human resource base for these countries, the numbers and knowledge diversity produced, still lack the capacity to optimally power research and development as well as supply the industrial biotechnology sectors of these countries. Realizing the need to address these issues at the grassroots level of higher education, Malaysia has taken an active step of bringing biotechnology into the classrooms of high schools throughout the country. These future generations of Malaysians, are hoped to progress towards manning and driving Malaysias BioValley initiatives (a biotech based R&D and industry cluster), towards the national dream of developed nation status by the year 2020, using biotechnology as an economic growth vehicle. Here, we share our experiences in developing and proliferating a biotechnology awareness program for Malaysian high schools. It is hoped that similar programs will strive towards similar objectives in other developing countries.

Crevicular alkaline phosphatase activity and rate of tooth movement of female orthodontic subjects under different continuous force applications

Purpose. This study is aimed to compare the effects of two different orthodontic forces on crevicular alkaline phosphatase activity, rate of tooth movement, and root resorption. Materials and Methods. Twelve female subjects of class II division 1 malocclusion participated. Maxillary canines with bonded fixed appliances acted as the tested teeth, while their antagonists with no appliances acted as the controls. Canine retraction was performed using nickel titanium coil spring that delivered forces of 100 gm or 150 gm to either side. Crevicular fluid was analyzed for ALP activity, and study models were casted to measure tooth movements. Root resorption was assessed using periapical radiographs before and after the force application. Results. ALP activity at the mesial sites peaked at week 1 for 150 gm group with significant differences when compared with the 100 gm group. Cumulative canine movements were significantly greater in the 150 gm force (2.10 ± 0.50 mm) than in the 100 gm force (1.57 ± 0.44 mm). No root resorption was in the maxillary canines after retraction. Conclusions. A force of 150 gm produced faster tooth movements and higher ALP activity compared with the 100 gm group and had no detrimental effects such as root resorption.

Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

Dental pulp tissue contains dental pulp stem cells (DPSCs). Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells) are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

Stem Cell Heterogeneity of Mononucleated Cells from Murine Peripheral Blood: Molecular Analysis

The main purpose of this paper was to determine the heterogeneity of primary isolated mononucleated cells that originated from the peripheral blood system by observing molecular markers. The isolated cells were cultured in complete medium for 4 to 7 days prior to the separation of different cell types, that is, adherent and suspension. Following a total culture time of 14 days, adherent cells activated the Cd105 gene while suspension cells activated the Sca-1 gene. Both progenitor markers, Cbfa-1 and Ostf-1, were inactivated in both suspension and adherent cells after 14-day culture compared to cells cultured 3 days in designated differentiation medium. In conclusion, molecular analyses showed that primary mononucleated cells are heterogeneous, consisting of hematopoietic stem cells (suspension) and mesenchymal stem cells (adherent) while both cells contained no progenitor cells.

Differentiation potential of human suspension mononucleated peripheral blood cells

Stem cells are known to have the ability to renew themselves and differentiate into a diverse range of specialized cell types. Currently, the differentiation potential of human peripheral blood mononucleated cells in suspension as stem cells is not well-understood. The aim of this study was to investigate the differentiation potential of suspension mononucleated cells from human peripheral blood to differentiate. The osteoblast and osteoclast differentiation potential of suspension peripheral blood mononucleated cells were examined by molecular, biochemical and cell morphology analyses. The expression of osteoblast marker (osteonectin, SPARC) and osteoclast marker (tartrate resistant acid phosphatase, TRAP) as well as high alkaline phosphate (ALP) and TRAP enzyme activity were observed at days 14 and 10 of osteoblast and osteoclast differentiation, respectively. Morphology analyses showed that mononucleated cells successfully differentiated into osteoblasts and osteoclasts. The existence of stem cells in mononucleated cells was evaluated by the expression of a stem cell factor (KIT) and a haematopoietic stem cell marker (signalling lymphocytic activation molecule family member 1, SLAMF1). This study has shown that these suspension mononucleated cells possess differentiation potential through in vitro study. Human peripheral blood suspension mononucleated cells that have multi-lineage differentiation potential may provide a new source of stem cells that may be used for bone regeneration and tissue engineering applications.

Stability of lactate dehydrogenase, aspartate aminotransferase, alkaline phosphatase and tartrate resistant acid phosphatase in human saliva and gingival crevicular fluid in the presence of protease inhibitor

The stability of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), tartrate resistant acid phos- phatase (TRAP) and alkaline phosphatase (ALP) activities from saliva and gingival crevicular fluid (GCF) with and with- out the addition of protease inhibitor (PI) at room temperature (RT; 25⁰C), 4°C and -20°C were investigated. AST, LDH, TRAP and ALP activities in saliva and GCF (n=9) with and without the addition of PI were assayed at 0 (control), 12, 24, 48, 72 h, one and two weeks. A paired t-test showed there were a significant differences (p<0.05) between LDH and TRAP activities in saliva, in the presence and without PI at all temperatures. ALP activity exhibited a significant difference in activity (p<0.05) in the presence and without PI at RT while no significant differences were observed at 4⁰C and -20⁰C. A significant difference (p<0.05) was observed in AST, LDH and TRAP activities (GCF) at RT and 4⁰C in the presence and without PI. We conclude that PI is essential for maintaining stable enzyme activities in saliva and GCF.