Cardioprotective potential of curcumin against norepinephrine‐induced cell death: a microscopic study

Cardiomyopathy and associated heart failure continues to be one of the most severe complications that threaten a large population. Curcumin, one of the three curcuminoids of the spice turmeric, is very well known for a multitude of health benefits and functions. Norepinephrine (NE), a catecholamine and also a stress hormone may cause the cardiomyocytes to develop increased sensitivity to death with its increasing concentrations. In this study, we investigated the cardioprotective effect of curcumin in NE‐induced cardiac apoptosis using several fluorescent and nonfluorescent microscopic techniques like DAPI, PI, Giemsa, PicroSirius and TUNEL. The aim of the study was to assess the effect of curcumin in preventing the occurrence of features underlying apoptosis such as nuclear disruption, chromatin condensation, DNA fragmentation and alterations in mitochondrial membrane permeability. Our results show that curcumin protects the cardiomyocytes against apoptosis significantly and also helps them to revert to their normal physiological state. Hence, we propose that curcumin has the potential to act as a therapeutic agent for the attenuation of NE‐induced cardiac cell death and modulation of apoptosis in H9c2 cardiomyocytes.     

Dynamic study of intramembranous particles in human fresh erythrocytes using an "in vitro cryotechnique"

For analyses of dynamic ultrastructures of erythrocyte intramembranous particles (IMPs) in situ, a quick-freezing method was used to stabilize the flow behavior of erythrocytes embedded in vitreous ice. Fresh human blood was jetted at various pressures through artificial tubes, in which the flowing erythrocytes were elongated from biconcave discoid shapes to elliptical ones, and quickly frozen in liquid isopentane-propane cryogen (-193 degrees C). They were freeze-fractured using a scalpel in liquid nitrogen, and routinely prepared for replica membranes. Many IMPs were observed on the protoplasmic freeze-fracture face (P-face) of the erythrocyte membranes. Some control erythrocytes under nonflowing or stationary conditions showed IMPs with their random distribution. However, other jetted erythrocytes under flowing conditions showed variously sized IMPs with much closer distribution. They were also arranged into parallel rows in some parts, and aggregated together. This quick-freezing method enabled for the first time the visualization of time-dependent topology and the molecular alteration of IMPs in dynamically flowing erythrocytes.     

An in vitro study to explore the role of prolylcarboxypeptidase in non-small cell lung cancer

Prolylcarboxypeptidase (PRCP) belongs to the S28 family of proteases, which is also a dipeptidyl peptidase.In this study, we demonstrate the expression pattern of PRCP in Non-small cell lung cancer (NSCLC). We found thatthe repression of PRCP expression by small interfering RNA successfully inhibited cell proliferation, migration, andinvasion. Further, we explored the involvement of PRCP in the regulation of epithelial-mesenchymal transition (EMT).The epithelial marker E-cadherin was significantly increased, meanwhile mesenchymal markers MUC1, vimentin, andSNAIL were markedly decreased in PRCP knockdown cells. Moreover, the downregulation of PRCP in the NSCLCcells induced the expression of apoptosis-related proteins in vitro. We performed RT-PCR in 30 pairs of clinical NSCLCtissues and adjacent non-cancerous tissues, which revealed significantly higher PRCP expression levels in cancer tissuesthan in adjacent non-cancerous tissues. Collectively the results from our study suggest a possible cancer promotion roleof PRCP in NSCLC.     

Surface activity of cancer cells: The fusion of two cell aggregates

A key feature that distinguishes cancer cells from all other cells is their capability to spread throughout the body. Although how cancer cells collectively migrate by following molecular rules which influence the state of cell-cell adhesion contacts has been comprehensively formulated, the impact of physical interactions on cell spreading remains less understood. Cumulative effects of physical interactions exist as the interplay between various physical parameters such as (1) tissue surface tension, (2) viscoelasticity caused by collective cell migration, and (3) solid stress accumulated in the cell aggregate core region. This review aims to point out the role of these physical parameters in cancer cell spreading by considering and comparing the rearrangement of various mono-cultured cancer and epithelial model systems such as the fusion of two cell aggregates. While epithelial cells undergo volumetric cell rearrangement driven by the tissue surface tension, which directs cell movement from the surface to the core region of two-aggregate systems, cancer cells rather perform surface cell rearrangement. Cancer cells migrate toward the surface of the two-aggregate system driven by the solid stress while the surface tension is significantly reduced. The solid stress, accumulated in the core region of the two-aggregate system, is capable of suppressing the movement of epithelial cells that can undergo the jamming state transition; however, this stress enhances the movement of cancer cells. We have focused here on the multi-scale rheological modeling approaches that aimed at reproducing and understanding these biological systems.     

Role of insulin-like growth factor binding protein-3 in breast cancer cell growth

The mitogenic effects of insulin-like growth factors (IGFs) are regulated by a family of insulin-like growth factor binding proteins (IGFBPs). One member of this family, IGFBP-3, mediates the growth-inhibitory and apoptosis-inducing effects of a number of growth factors and hormones such as transforming growth factor-beta, retinoic acid, and 1,25-dihydroxyvitamin D3. IGFBP-3 may act in an IGF-dependent manner by attenuating the interaction of pericellular IGFs with the type-I IGF receptor. It may also act in an IGF-independent manner by initiating intracellular signaling from a cell surface receptor, or by direct nuclear action, or both. The possibility of a membrane-bound receptor is strengthened by recent studies which have identified members of the transforming growth factor-beta receptor family as having a role, either directly or indirectly, in signaling from the cell surface by IGFBP-3. A number of growth factors and hormones stimulate the expression and secretion of cellular IGFBP-3, which then signals from the cell surface to bring about some of the effects attributed to the primary agents. Within the cell, the apoptosis-inducing tumor suppressor, p53, can also induce IGFBP-3 expression and secretion. Since IGFBP-3 upregulates the cell cycle inhibitor, p21(Waf1), and increases the ratio of proapoptotic to antiapoptotic members of the Bcl family, it appears to exert the same effects on major downstream targets of cell signaling as p53 does. The nuclear localization of IGFBP-3 has been described in a number of cell types. IGFBP-3 may act to import IGFs or other nuclear localization signal-deficient signaling molecules into the nucleus. It may also act directly in the nucleus by enhancing the activity of retinoid X receptor-alpha and thereby promote apoptosis. All of the above phenomena will be discussed with particular emphasis on the growth of breast cancer cells.     

Interface biomechanics of the Anca Dual fit hip stem: an in vitro experimental study

The Anca Dual Fit hip stem (Cremascoli Wright, Milan, Italy) is a partially cemented stem developed to overcome the drawbacks of both cemented and uncemented fixations. Its design was based on the hypothesis that partial cementing would ensure the primary stability necessary to allow bone ingrowth on the cement-free stem surfaces. At the same time, the limitation of the cement to the proximal regions would prevent stress-shielding by increasing proximal load transfer. After finite element (FE) simulations and in vitro primary stability assessment, an analysis of the long-term stability of the Anca Dual Fit stem was necessary to conclude the preclinical testing. Three stems were implanted in composite femurs and subjected to testing for 1 x 10(6) cycles, each cycle reproducing the activity of stair climbing. The simulation was designed so as to replicate the physiological loading in a simplified, yet relevant way for this test. Various measurements were collected before, during and after the test in order to give exhaustive information on the response of the implant to long-term, cyclic loading. The present study confirmed the positive results of previous investigations, and proved that the Anca Dual Fit stem has excellent long-term stability; therefore successful clinical outcomes are predicted.     

In vitro and in silico (IVIS) flow characterization in an idealized human airway geometry using laser Doppler anemometry and computational fluid dynamics techniques

The air flow in idealized human airway geometry was studied using computational and experimental methods. A computational fluid dynamics model developed to determine the air flow characteristics in airways was validated by comparison of the experimental velocity profiles obtained with laser Doppler anemometric measurements with numerical data. A good correlation was found between the values obtained with the two methods. Both the measurements and the calculations showed the flow to be laminar in the trachea region of the airway model, but it is affected by the airway geometry in subsequent airways.     

Overexpression of inhibin α (1-32) fusion protein promotes apoptosis and cell cycle arrest in a cervical cancer cell model (Hela cells)

Inhibins play important roles in the reproductive system. To evaluate whether inhibin α (1-32) fusion protein plays a role in cervical cancer growth, the plasmid pVAX-inhα was constructed and its effect on proliferation and apoptosis of the human cervical cancer cell line (Hela) was checked by flow cytometry and real-time PCR. The expression and localization of inhibin α protein were detected by RT-PCR and confocal microscopy which showed that inhibin α protein was expressed and localized in the nucleus of Hela cells. Over expression of inhibin α gene significantly induced cell apoptosis and ceased S phase of cell cycle. Furthermore, cell proliferation was significantly suppressed 96 h post-transfection and mRNA level of anti-apoptosis genes (Bcl-2, NFκB) were decreased but pro-apoptosis genes (Bax, wild type p53) and inhibin co receptor (TGFβR3) were increased, indicating that inhibin, through its co-receptor, might activate apoptotic and cell growth cascades which regulate proliferation and apoptosis in Hela cells. These results suggest that inhibin α (1-32) fusion protein, located in the cell nucleus, can regulate Hela cells growth and apoptosis by induction of apoptotic pathways such as NFκB, Bcl-2 and p53 families. These findings may have a significant impact on future research regarding cervical cancer cell lines     

Morphological color changes in fish: regulation of pigment cell density and morphology

Pigment cells enable fish to change their coloration. It has been recognized that fish color changes can be divided into two categories; one is a physiological color change, which is attributed to rapid motile responses of chromatophores, and the other is a morphological color change, which results from changes in the morphology and density of chromatophores. Long-term adaptation of fish to a certain background can be a general cue to morphological color changes, and has been studied from the beginning of the 19th century. Although the motile mechanism and its control in fish chromatophores are now being elucidated, it is not yet clear how chromatophores change their density and what controls morphological color changes. In recent years, chromatophores, especially melanophores, have been shown to differentiate and to die by apoptosis under the influence of factors that regulate motile responses. Those factors are likely to utilize common intracellular signaling pathways used in part to regulate both types of color changes. In this article, after briefly reviewing the history of early studies, recent findings are discussed relevant to increases or decreases in chromatophores, and changes in their morphology. Finally, morphological color changes are discussed as physiological phenomena involved in the balance between differentiation and apoptosis of chromatophores.     

The toxic effect of ketamine on SH‐SY5Y neuroblastoma cell line and human neuron

Ketamine used as an injectable anesthetic in human and animal medicine is also a recreational drug used primarily by young adults often at all night dance parties in nightclubs. The percentage of ketamine users has grown very fast in the last 5 years worldwide. However, this leads to the serious question of the long‐term adverse effects of ketamine on our nervous system, particularly the brain, because ketamine as an NMDA antagonist could cause neurons to commit apoptosis. Our study therefore aimed to find out the chronic effect of ketamine on neuron using prolonged incubation (48 h) of neuronal cells with ketamine in culture. Our results showed that differentiated neuronal cells were prone to the toxicity of ketamine but probably less susceptible than undifferentiated neuronal cells and fibroblasts. This suggested that the ketamine abuse would be harmful to many other organs as well as the brain. Our results also confirmed that the toxicity of ketamine is related to apoptosis via the Bax/Bcl‐2 ratio pathway and caspase‐3 in the differentiated neuronal cells. Therefore, long‐term ketamine treated cell or animal models should be sought to study this multiorgan effects of ketamine. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Influence of estradiol analogue on cell growth,morphology and death in esophageal carcinoma cells

2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0μM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4μM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4μM 2-methoxyestradiol-bis-sulphamate.     

Arsenic trioxide inhibits the activity of SphK1 by decreasing the level of phosphatidylserine and phosphatidic acid in the human gastric cancer cell line MGC-803

Sphingosine kinase 1 (SphK1) is an important synthetase during the synthesis of sphingosine-1-phosphate (S1P)from sphingosine (Sph). Previous studies demonstrated that arsenic trioxide (As₂O₃) could reduce the level of S1P in humangastric cancer cell line MGC-803, indicating that As₂O₃ may inhibit the activity of SphK1. In this study, the effect of As₂O₃on the SphK1 activation pathway was investigated. Western blot and quantitative real-time PCR analysis were used toevaluate the changes in protein and mRNA levels. The multi-dimensional mass spectrometry-based shotgun lipidomicsmethod (MDMS-SL) was used for the quantitative detection of phosphatidylserine (PS) and phosphatidic acid (PA). Theresults revealed that As₂O₃ did not affect the protein and mRNA expression of SphK1 in the MGC-803 cells. However,As₂O₃ increased the levels of p-ERK1/2 and CIB1 in the SphK1 activation pathway and decreased the levels of PS andPA in the MGC-803 cells. The outcomes suggested that As₂O₃ may enhance the activity of SphK1 by increasing thelevels of p-ERK1/2 and CIB1 and decrease the activity of SphK1 by decreasing the levels of PS and PA. It was suggestedthat the inhibition effect is stronger and resulting in an overall decrease in the activity of SphK1.     

Reflectance in situ hybridization (RISH): detection,by confocal reflectance laser microscopy,of gold-labelled riboprobes in breast cancer cell lines and histological specimens

A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1-nm colloidal-gold particles after RNA/RNA in situ hybridization, using digoxigenin-labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras-related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three-dimensional imaging were used for visualization. A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.     

Entamoeba histolyticaelectrondense granules secretion in vitro and in vivo: ultrastructural study

Electron dense granules (EDGs) were identified by transmission electron microscopy in Entamoeba histolytica trophozoites recovered from hamster liver lesions. Abundant granules were present in trophozoites recovered after 15 min of liver inoculation. Variation in the size and morphology of these EDGs was also observed. Numerous granules were present in the plasma membrane when these parasites were incubated for 5 min with MDCK monolayers. Release of these EDGs was suggested by the presence of granules in contact with the surface of the target cell plasma membrane. Parasite phagocytic invaginations were observed after 10 min of parasite-monolayer interaction. In these structures, scarce granules were seen. Granules secretion was corroborated by obtaining of a pellet of these small structures from the incubation of trophozoites with collagen supernatant. Collagenase and gellatinase activity of this pellet was identified in SDS-PAGE gels. EDGs were also present in amebic hamster liver lesions. Our observations corroborate that these granules are secreted and suggest that may participate in the cytopathic effect of E. histolytica both in vitro and in vivo.     

Prediction of calcium concentration in human blood serum using an artificial neural network

A predictive method, based on artificial neural network (ANN) has been developed to study absorbance and pH effects on the equilibrium of blood serum. This strategy has been used to analyze serum samples and to predict the calcium concentration in blood serum. A dedicated data acquisition system is designed and fabricated using a LPC2106 microcontroller with light emitting diode (LED) as source and photodiode as sensor to measure absorbance and to calculate the calcium concentration. A multilayer neural network with back propagation (BP) training algorithm is used to simulate different concentration of calcium (Ca²⁺) as a function of absorbance and pH, to correlate and predict calcium concentration. The computed calcium concentration by neural network is quite satisfactory with correlations R² = 0.998 and 0.995, standard errors of 0.0127 and 0.0122 in validation and testing stages respectively. Statistical analysis are carried out to check the accuracy and precision of the proposed ANN model and validation of results produce a relative error of about 3%. These results suggest that ANN can be efficiently applied and is in good agreement with values obtained with the current clinical spectrophotometric methods. Hence, ANN can be used as a complementary tool for studying metal ion complexion, with special attention to the blood serum analysis.