Instrumental development attachable to high magnification microscopes for obtaining totally focalized images

Betalains are water-soluble nitrogenous vacuolar pigments present in flowers and fruits of many caryophyllales with potent antioxidant properties. In the present study the antiproliferative effects of betanin, a principle betacyanin pigment, isolated from the fruits of Opuntia ficus-indica, was evaluated on human chronic myeloid leukemia cell line (K562). The results show dose and time dependent decrease in the proliferation of K562 cells treated with betanin with an IC(50) of 40 microM. Further studies involving scanning and transmission electron microscopy revealed the apoptotic characteristics such as chromatin condensation, cell shrinkage and membrane blebbing. Agarose electrophoresis of genomic DNA of cells treated with betanin showed fragmentation pattern typical for apoptotic cells. Flow cytometric analysis of cells treated with 40 microM betanin showed 28.4% of cells in sub G0/G1 phase. Betanin treatment to the cells also induced the release of cytochrome c into the cytosol, poly (ADP) ribose polymerase (PARP) cleavage, down regulation Bcl-2, and reduction in the membrane potentials. Confocal microscopic studies on the cells treated with betanin suggest the entry of betanin into the cells. These studies thus demonstrate that betanin induces apoptosis in K562 cells through the intrinsic pathway and is mediated by the release of cytochrome c from mitochondria into the cytosol, and PARP cleavage. The antiproliferative effects of betanin add further value to the nutritional characteristics of the fruits of O. ficus-indica.

An automated fluorescence method for the detection of neuronal cell death by necrosis and apoptosis with sequential acridine orange (AO) and ethidium bromide (EB) staining using confocal microscopy is described. Since cell nuclei during apoptosis become acidic, AO staining was utilized to distinguish live neurons from neurons undergoing apoptosis, using the AO property to shift its fluorescence from green at normal pH toward brilliant orange-red in the process of acidification. Further EB application labels nuclei of necrotic neurons in red. Sequential treatment by AO and EB can be employed as an express vitality test to count fractions of live and dead cell via apoptosis and necrosis, respectively. An algorithm of automatic quantification of cell types is based on the image correlation analysis. Our conclusion is validated by experiments with the vital dye trypan blue and the pharmacological study of receptor subtypes involved in the excitotoxicity. The approach described here, therefore, offers an express, easy, sensitive and reproducible method by which necrosis and apoptosis can be recognized and quantified in a population of living neurons. Because this assay does not require any preliminary tissue treatment, fixation or dissociation in a cell suspension its utility is likely to be extended for measuring cell viability and cytotoxicity on a variety of living preparations (tissues, brain slices and cell cultures).

The current rapid progression in tissue engineering and local gene delivery systems has enhanced applications of osseointegration in dental implants. In this study, porous chitosan/collagen scaffolds were prepared through a freeze-drying process, and loaded with an adenoviral vector encoding human bone morphogenetic proteins (BMP7). These scaffolds were evaluated in vitro by scanning electron microscopy (SEM), and human periodontal ligament cells (HPLCs) were seeded in this scaffold. We used Reverse transcription-polymerase chain reaction (RT-PCR) to determine the expression levels of osteopontin and bone sialoprotein. Alkaline phosphatase (ALP) activity was also determined. Then these scaffolds were implanted into defects on both sides of the mandible. Three months later, the animals were sacrificed and non-decalcified sections were evaluated histologically. Histomorphometric analyses were performed at the bone-implant interface using the image obtained by confocal laser scanning microscopy. Results indicated that the scaffold containing Ad-BMP7 exhibited the higher ALP activity, and the expression of osteopontin and bone sialoprotein were up-regulated. After implanting in defects around implant, the bone formation in Ad-BMP7 scaffolds was greater than that in other scaffolds at 4 or 8 weeks. This study demonstrated the potential of chitosan/collagen scaffold combined Ad-BMP7 as a good substrate candidate in bone tissue engineering.