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Serum Deprivation (serum + deprivation)

Distribution by Scientific Domains

Medical Sciences	50%
Life Sciences	45%

Selected Abstracts

Effect of Neural Stem Cells on Apoptosis of PC12 Cells Induced by Serum Deprivation

BIOTECHNOLOGY PROGRESS, Issue 4 2007
Xiangqin Li
Neural stem cells (NSCs) have a bright application prospect to be used to treat neurodegenerative diseases due to their capacity to give rise to the appropriate cell types when they are grafted. At present, however, the function of NSCs after transplantation is not quite ensured, whether to replace the degenerative cells or to secrete nutrient factors. On the other hand, pheochromocytoma cell line 12 (PC12) cells have been widely used for investigating Parkinsonapos;s disease (PD) since their apoptosis is similar to that of dopaminergic neuron cells. Therefore, the possible cytoprotective effects of NSCs on the apoptosis of PC12 cells induced by serum deprivation were investigated in this paper. PC12 cells were cocultured with NSCs in DMEM/F12 medium free of serum, and their morphologies, viabilities, and survival were observed with an inverted microscope and assessed with a CCK-8 assay. In addition, the concentrations of glial derived neurotrophic factor (GDNF) in different medium were detected with a GDNF Elisa kit, and the mechanism of NSCapos;s protective effect on PC12 cell apoptosis induced by serum deprivation was analyzed. The results showed that (1) PC12 cell apoptosis induced by serum deprivation increased with time, and only about 44.25% PC12 cells survived after 72 h; (2) NSCs culture medium protected against PC12 cell apoptosis insignificantly; (3) NSCs' supernatant and NSCs mildly prevented PC12 cells from apoptosis; (4) the amount of GDNF secreted by NSCs increased after the coculture with the apoptotic PC12 cells induced by serum deprivation. It can be concluded that there exists clear interaction between NSCs and apoptotic PC12 cells, and that GDNF secretion from NSCs is one of the important mechanisms to prevent the apoptosis of PC12 cells. [source]

Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+ and loss of NAD+

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2010
Christian T. Sheline
Abstract Trophic deprivation-mediated neuronal death is important during development, after acute brain or nerve trauma, and in neurodegeneration. Serum deprivation (SD) approximates trophic deprivation in vitro, and an in vivo model is provided by neuronal death in the mouse dorsal lateral geniculate nucleus (LGNd) after ablation of the visual cortex (VCA). Oxidant-induced intracellular Zn2+ release ([Zn2+]i) from metallothionein-3 (MT-III), mitochondria or ,protein Zn2+', was implicated in trophic deprivation neurotoxicity. We have previously shown that neurotoxicity of extracellular Zn2+ required entry, increased [Zn2+]i, and reduction of NAD+ and ATP levels causing inhibition of glycolysis and cellular metabolism. Exogenous NAD+ and sirtuin inhibition attenuated Zn2+ neurotoxicity. Here we show that: (1) Zn2+ is released intracellularly after oxidant and SD injuries, and that sensitivity to these injuries is proportional to neuronal Zn2+ content; (2) NAD+ loss is involved , restoration of NAD+ using exogenous NAD+, pyruvate or nicotinamide attenuated these injuries, and potentiation of NAD+ loss potentiated injury; (3) neurons from genetically modified mouse strains which reduce intracellular Zn2+ content (MT-III knockout), reduce NAD+ catabolism (PARP-1 knockout) or increase expression of an NAD+ synthetic enzyme (Wlds) each had attenuated SD and oxidant neurotoxicities; (4) sirtuin inhibitors attenuated and sirtuin activators potentiated these neurotoxicities; (5) visual cortex ablation (VCA) induces Zn2+ staining and death only in ipsilateral LGNd neurons, and a 1 mg/kg Zn2+ diet attenuated injury; and finally (6) NAD+ synthesis and levels are involved given that LGNd neuronal death after VCA was dramatically reduced in Wlds animals, and by intraperitoneal pyr vate or nicotinamide. Zn2+ toxicity is involved in serum and trophic deprivation-induced neuronal death. [source]

,-Arrestin 2 regulates toll-like receptor 4-mediated apoptotic signalling through glycogen synthase kinase-3,

IMMUNOLOGY, Issue 4 2010
Hui Li
Summary Toll-like receptor 4 (TLR4), a key member of the TLR family, has been well characterized by its function in the induction of inflammatory products of innate immunity. However, the involvement of TLR4 in a variety of apoptotic events by an unknown mechanism has been the focus of great interest. Our investigation found that TLR4 promoted apoptotic signalling by affecting the glycogen synthase kinase-3, (GSK-3,) pathway in a serum-deprivation-induced apoptotic paradigm. Serum deprivation induces GSK-3, activation in a pathway that leads to subsequent cell apoptosis. Intriguingly, this apoptotic cascade is amplified in presence of TLR4 but greatly attenuated by ,-arrestin 2, another critical molecule implicated in TLR4-mediated immune responses. Our data suggest that the association of ,-arrestin 2 with GSK-3, contributes to the stabilization of phospho-GSK-3,, an inactive form of GSK-3,. It becomes a critical determinant for the attenuation of TLR4-initiated apoptosis by ,-arrestin 2. Taken together, we demonstrate that the TLR4 possesses the capability of accelerating GSK-3, activation thereby deteriorating serum-deprivation-induced apoptosis; ,-arrestin 2 represents an inhibitory effect on the TLR4-mediated apoptotic cascade, through controlling the homeostasis of activation and inactivation of GSK-3,. [source]

Are mesenchymal stromal cells from children resistant to apoptosis?

CELL PROLIFERATION, Issue 3 2009
H. Dimitriou
Objectives:, Mesenchymal stromal cells (MSC) represent a novel cellular candidate in the field of transplantation and tissue regeneration. Their clinical application requires their in vitro expansion. The aim of this study was to assess the effect of conditions that would favour apoptosis, and of long-term expansion, on the characteristics of MSC from children. Materials and methods:, Bone marrow mononuclear cells were cultured for 10 passages (P1,P10). Expression of CD105, CD146, CD95 and apoptosis by 7-amino-actinomycin D staining were evaluated. CFU-F and cell doubling time (DT) were assessed in every passage. Cell-cycle study was performed at P2 and P6. Results:, CFU-F decreased from 38 ± 3.7 at P2 to 9.6 ± 3.2 per 10 MSC/cm2 at P10 and DT increased from 1.93 ± 0.1 (P2) to 6.1 ± 2.45 days (P10). A low percentage of apoptotic (dead) cells was detected at P2 and this did not change until P10. Cells at P2 were at G0/G1 phase, but in advanced passages more cells were in an active state. Induction of apoptosis (addition of anti-Fas agonist antibody) using standard culture conditions, showed a minor effect on MSC survival. Serum deprivation of MSC (up to 72 h) revealed no substantial apoptotic effect while cells retained their tri-lineage differentiation capacity. Conclusions:, We conclude that MSC from children retain their functional characteristics throughout serial passages and remain stable under conditions that usually cause apoptosis. These features render MSC, especially those of early passages, optimal candidates for use in clinical applications. [source]

Leptin protects cardiomyocytes from serum-deprivation-induced apoptosis by increasing anti-oxidant defence

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 10 2010
Juan Zheng
Summary 1. Leptin, an important adipose-derived hormone, can be associated with cardiac pathophysiology; however, the role of leptin in cardiomyocyte apoptosis is poorly understood. The present study examines serum-deprivation-induced apoptosis in primary cultured cardiomyocytes treated with leptin. 2. Cardiomyocytes were subjected to serum deprivation in the presence or absence of leptin (5 or 50 nmol/L) for 48 h. Apoptosis was determined by Hoechst 33258 and Annexin V-FITC/propidium iodide dual staining. Cell viability, malondialdehyde (MDA) content, caspase 3 activation, and the expression and enzyme activity of superoxide dismutase (SOD) were measured. Small interference RNA (siRNA) targeting SOD1 and SOD2 were used to knockdown their expression and measure apoptosis. 3. Serum deprivation caused nearly 30% of apoptosis in cardiomyocytes, and an approximately 60% decrease in cell viability. The mRNA levels and the activated form of caspase 3 were greatly increased. In the presence of leptin, the apoptotic rate was reduced to approximately 15%, cell viability was increased and the activation of caspase 3 was partially inhibited. Additionally, the augmented lipid peroxidation (MDA formation) was abolished, and the impaired activities of SOD1 and SOD2 were restored by leptin. The mRNA expression of SOD2, but not SOD1, was stimulated by leptin. Transfection with siRNA that cause deficiency of either SOD1 or SOD2 attenuated the anti-apoptotic effects of leptin. 4. The results suggest that leptin inhibits serum-deprivation-induced apoptosis in cardiomyocytes by activating SOD. The present study outlines the direct actions of leptin in cardiac disorders that are related to elevated leptin levels. [source]

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

FEBS JOURNAL, Issue 8 2007
Patryk Krzemi
We characterized the expression and functional properties of the ADP-sensitive P2Y1 and P2Y12 nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y12 receptor relative to P2Y1 was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y1 receptor was low, and the P2Y12 receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y12 receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y12 receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y1 receptor, indicating the inhibitory role of P2Y1 in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y1 to P2Y12 would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation. [source]

Upregulated claudin-1 expression confers resistance to cell death of nasopharyngeal carcinoma cells

INTERNATIONAL JOURNAL OF CANCER, Issue 6 2010
Jeng-Woei Lee
Abstract Accumulating evidence reveals that aberrant expression of claudins manifests in various tumors; however, their biological functions are poorly understood. Here, we report on the elevated expression of claudin-1 in nasopharyngeal carcinoma (NPC) cell lines under serum deprivation or fluorouracil (5-FU) treatment. Interestingly, an increase in expression of claudin-1 considerably reduced apoptosis rather than enhancing cell proliferation. However, claudin-1 expression and activity were unaffected by external stimuli or Akt and NF-,B activation. Notably, predominant cytoplasmic and nuclear localization of claudin-1 in NPC cells reflected the aforementioned feature. On the other hand, loss of epithelial morphology and E-cadherin expression was associated with serum withdrawal in NPC cells. Interestingly, restoration of E-cadherin inhibited the protein elevation and antiapoptotic activity of claudin-1. In conclusion, our data demonstrate the regulation and novel biological function of claudin-1 and indicate the important role of claudin-1 in NPC tumorigenesis. [source]

Importance of C16 ceramide accumulation during apoptosis in prostate cancer cells

INTERNATIONAL JOURNAL OF UROLOGY, Issue 2 2006
MASATOSHI ETO
Aim:, Adenocarcinoma of the prostate is one of the most frequently diagnosed non-cutaneous cancers and the second leading cause of cancer-related deaths among men in the United States. To fully understand the role of ceramide during apoptosis induced by androgen ablation, we modified the levels of intracellular ceramide by pharmacological agents as well as through serum deprivation in androgen-dependent and independent cell lines. Methods:, Ceramide levels were modified using N-oleoylethanolamine (NOE), sphingosine-1-phosphate (S1P) as well as through serum deprivation, in LNCaP, DU145 and PC-3 prostate cancer cells. Various methods including nonyl acridine orange staining, propidium iodide staining/cell cycle analysis and lipid analysis were utilized. Results:, Our results demonstrate that the inhibition of acid ceramidase by NOE enhances the intracellular ceramide levels induced by androgen ablation in androgen-dependent LNCaP cells, and is accompanied by an increase in apoptotic cells. Sphingosine 1-phosphate had no effect in rescuing LNCaP cells from apoptosis induced by androgen ablation. Our results also show that serum deprivation causes intracellular ceramide accumulation and apoptosis in androgen-independent prostate cancer cells. Conclusions:, Our studies indicate that the increase in intracellular ceramide itself, but not the balance between ceramide and S1P, determines whether LNCaP cells undergo apoptosis. Our results also show that the increase in intracellular ceramide strongly correlates with apoptosis induced by serum deprivation even in androgen-independent prostate cancer cell lines. [source]

Expression of FGFR3 with the G380R Achondroplasia Mutation Inhibits Proliferation and Maturation of CFK2 Chondrocytic Cells

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000
Janet E. Henderson
Abstract A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression. [source]

Human islet-derived precursor cells can cycle between epithelial clusters and mesenchymal phenotypes

JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009
Behrous Davani
Abstract We showed previously that undifferentiated, proliferating human islet-derived precursor cells (hIPCs) are a type of mesenchymal stem/stromal cell (MSC) that can be induced by serum deprivation to form clusters and ultimately differentiate in vitro to endocrine cells. We also demonstrated that partially differentiated hIPC clusters, when implanted under the kidney capsules of mice, continued to differentiate in vivo into hormone-producing cells. However, we noted that not all hIPC preparations yielded insulin-secreting cells in vivo and that in some animals no hormone-expressing cells were found. This suggested that the implanted cells were not always irreversibly committed to further differentiation and may even de-differentiate to a mesenchymal phenotype. In this study, we show that human cells with a mesenchymal phenotype are indeed found in the grafts of mice implanted with hIPCs in epithelial cell clusters (ECCs), which are obtained after 4-day in vitro culture of hIPCs in serum-free medium (SFM); mesenchymal cells were predominant in some grafts. We could mimic the transition of ECCs to de-differentiated mesenchymal cells in vitro by exposure to foetal bovine serum (FBS) or mouse serums, and to a significantly lesser extent to human serum. In a complementary series of experiments, we show that mouse serum and FBS are more effective stimulants of mesenchymal hIPC migration than is human serum. We found that proliferation was not needed for the transition from ECCs to de-differentiated cells because mitomycin-treated hIPCs that could not proliferate underwent a similar transition. Lastly, we show that cells exhibiting a mesenchymal phenotype can be found in grafts of adult human islets in mice. We conclude that epithelial-to-mesenchymal transition (EMT) of cells in hIPC ECCs can occur following implantation in mice. This potential for EMT of human islets or differentiated precursor cells must be considered in strategies for cell replacement therapy for diabetes. [source]

Supersensitivity of P2X7 receptors in cerebrocortical cell cultures after in vitro ischemia

JOURNAL OF NEUROCHEMISTRY, Issue 5 2005
Kerstin Wirkner
Abstract Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2,-3,- O -(4-benzoylbenzoyl)-adenosine 5,-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways. [source]

Endogenous and Exogenous Fibroblast Growth Factor 2 Support Survival of Chick Retinal Neurons by Control of Neuronal Neuronal bcl-xL and bcl-2 Expression Through a Fibroblast Berowth Factor Receptor 1- and Erk-Dependent Pathway

JOURNAL OF NEUROCHEMISTRY, Issue 1 2000
Laurent Désiré
Abstract : Fibroblast growth factor (FGF) 2 is a survival factor for various cell types, including retinal neurons. However, little is understood about the molecular bases of the neuroprotective role of FGF2 in the retina. In this report, FGF2 survival activity was studied in chick retinal neurons subjected to apoptosis by serum deprivation. Exogenous FGF2 supported neuronal survival after serum deprivation and increased neuronal bcl-xL and bcl-2 expression, through binding to its receptor R1 (FGF-R1), and subsequent extracellular signal-regulated kinase (ERK) activation. Endogenous FGF2 was transiently overexpressed after serum deprivation. Its down-regulation by antisense oligonucleotides and blockade of its signaling pathway (binding to FGF-R1, tyrosine phosphorylation, and ERK inhibition) decreased bcl-xL and bcl-2 levels and and enhanced apoptosis, suggesting that endogenous FGF2 supported neuronal survival through a pathway similar to that of exogenous FGF2. This pathway may serve to up-regulate, or maintain, bcl-xL and bcl-2 levels that normally decrease during the onset of apoptosis. Indeed, long-term ERK activation and high bcl-xL levels are necessary for the survival activity of both exogenous and endogenous FGF2. Because FGF2 is upregulated following retinal injury in vivo, we suggest that an injury-stimulated autocrine/paracrine FGF2 loop may serve to maintain high levels of survival proteins, such as Bcl-xL, through ERK activation in retinal neurons. [source]

Effect of transforming growth factor-beta1 on expression of the connective tissue growth factor (CCN2/CTGF) gene in normal human gingival fibroblasts and periodontal ligament cells

JOURNAL OF PERIODONTAL RESEARCH, Issue 2 2009
H. Takeuchi
Background and Objective:, Connective tissue growth factor (CCN2/CTGF) plays an important role in wound healing and regulation of the extracellular matrix in periodontal tissue. However, the functional relationship between altered transforming growth factor-beta1 levels and CCN2/CTGF has not been extensively investigated in human gingival fibroblasts and periodontal ligament cells. This study investigated the effects of transforming growth factor-beta1 on the expression of the CCN2/CTGF gene in human gingival fibroblasts and periodontal ligament cells in vitro. Material and Methods:, Cells were isolated from normal periodontal tissues and cultured in Dulbecco's modified Eagle's minimal essential medium/F12 containing 10% fetal bovine serum. Subconfluent cells were maintained under serum deprivation for 24 h then treated with Dulbecco's modified Eagle's minimal essential medium/F12 containing 0.5% fetal bovine serum (control) and 0.1, 1, 5 or 10 ng/mL of transforming growth factor-beta1 for 24, 48 or 72 h. The effects of transforming growth factor-beta1 on CCN2/CTGF mRNA expression were measured by reverse transcription,polymerase chain reaction. CCN2/CTGF protein was quantitatively analyzed using enzyme-liked immunosorbent assay. Subcellular distribution of CCN2/CTGF protein in both human gingival fibroblasts and periodontal ligament cells was observed using immunofluorescence microscopy. Results:, In both human gingival fibroblasts and periodontal ligament cells, the expression of CCN2/CTGF mRNA and CCN2/CTGF protein was significantly increased, in a dose- and time-dependent manner, in the presence of transforming growth factor-beta1. Moreover, immunofluorescence analysis indicated that immunoreactivity to CCN2/CTGF showed a granular pattern of protein localization. Conclusion:, The expression of CCN2/CTGF mRNA and protein was induced by transforming growth factor-beta1 in human gingival fibroblasts and periodontal ligament cells. These results suggest that CCN2/CTGF plays an important role in wound healing and in the regeneration of periodontal tissue. [source]

Astaxanthin, a dietary carotenoid, protects retinal cells against oxidative stress in-vitro and in mice in-vivo

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2008
Yoshimi Nakajima
We have investigated whether astaxanthin exerted neuroprotective effects in retinal ganglion cells in-vitro and in-vivo. In-vitro, retinal damage was induced by 24-h hydrogen peroxide (H2O2) exposure or serum deprivation, and cell viability was measured using a WST assay. In cultured retinal ganglion cells (RGC-5, a rat ganglion cell-line transformed using E1A virus), astaxanthin inhibited the neurotoxicity induced by H2O2 or serum deprivation, and reduced the intracellular oxidation induced by various reactive oxygen species (ROS). Furthermore, astaxanthin decreased the radical generation induced by serum deprivation in RGC-5. In mice in-vivo, astaxanthin (100 mg kg,1, p.o., four times) reduced the retinal damage (a decrease in retinal ganglion cells and in thickness of inner plexiform layer) induced by intravitreal N -methyl- d -aspartate (NMDA) injection. Furthermore, astaxanthin reduced the expressions of 4-hydroxy-2-nonenal (4-HNE)-modified protein (indicator of lipid peroxidation) and 8-hydroxy-deoxyguanosine (8-OHdG; indicator of oxidative DNA damage). These findings indicated that astaxanthin had neuroprotective effects against retinal damage in-vitro and in-vivo, and that its protective effects may have been partly mediated via its antioxidant effects. [source]

The liver stage of Plasmodium berghei inhibits host cell apoptosis

MOLECULAR MICROBIOLOGY, Issue 3 2005
Claudia Van De Sand
Summary Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite,host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei -infected cells and compared their level of expression and their distribution to that of non-infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non-infected HepG2 cells, whereas P. berghei -infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)-,/d -galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non-treated control mice as well as in TNF-,-treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death. [source]

Retroviral vector production under serum deprivation: The role of lipids

BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009
A.F. Rodrigues
Abstract The use of retroviral vectors for gene therapy applications demands high titer preparations and stringent quality standards. However, the manufacturing of these vectors still represents a highly challenging task due to the low productivity of the cell lines and reduced stability of the vector infectivity, particularly under serum-free conditions. With the objective of understanding the major limitations of retroviral vector production under serum deprivation, a thorough study of viral production kinetics, vector characterization and cell growth and metabolic behavior was conducted, for 293 FLEX 18 and Te Fly Ga 18 producer cell lines using different serum concentrations. The reduction of serum supplementation in the culture medium resulted in pronounced decreases in cell productivity of infectious vector, up to ninefold in 293 FLEX 18 cells and sevenfold in Te Fly Ga 18 cells. Total particles productivity was maintained, as assessed by measuring viral RNA; therefore, the decrease in infectious vector production could be attributed to higher defective particles output. The absence of the serum lipid fraction was found to be the major cause for this decrease in cell viral productivity. The use of delipidated serum confirmed the requirement of serum lipids, particularly cholesterol, as its supplementation not only allowed the total recovery of viral titers as well as additional production increments in both cell lines when comparing with the standard 10% (v/v) FBS supplementation. This work identified lower production ratios of infectious particles/total particles as the main restraint of retroviral vector production under serum deprivation; this is of the utmost importance concerning the clinical efficacy of the viral preparations. Lipids were confirmed as the key serum component correlated with the production of infective retroviral vectors and this knowledge can be used to efficiently design medium supplementation strategies for serum-free production. Biotechnol. Bioeng. 2009; 104: 1171,1181. © 2009 Wiley Periodicals, Inc. [source]

Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene

BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006
Shin Sik Choi
Abstract It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein. © 2006 Wiley Periodicals, Inc. [source]

Effect of Neural Stem Cells on Apoptosis of PC12 Cells Induced by Serum Deprivation

Piracetam improves mitochondrial dysfunction following oxidative stress

BRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2006
Uta Keil
Mitochondrial dysfunction including decrease of mitochondrial membrane potential and reduced ATP production represents a common final pathway of many conditions associated with oxidative stress, for example, hypoxia, hypoglycemia, and aging. Since the cognition-improving effects of the standard nootropic piracetam are usually more pronounced under such pathological conditions and young healthy animals usually benefit little by piracetam, the effect of piracetam on mitochondrial dysfunction following oxidative stress was investigated using PC12 cells and dissociated brain cells of animals treated with piracetam. Piracetam treatment at concentrations between 100 and 1000 ,M improved mitochondrial membrane potential and ATP production of PC12 cells following oxidative stress induced by sodium nitroprusside (SNP) and serum deprivation. Under conditions of mild serum deprivation, piracetam (500 ,M) induced a nearly complete recovery of mitochondrial membrane potential and ATP levels. Piracetam also reduced caspase 9 activity after SNP treatment. Piracetam treatment (100,500 mg kg,1 daily) of mice was also associated with improved mitochondrial function in dissociated brain cells. Significant improvement was mainly seen in aged animals and only less in young animals. Moreover, the same treatment reduced antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in aged mouse brain only, which are elevated as an adaptive response to the increased oxidative stress with aging. In conclusion, therapeutically relevant in vitro and in vivo concentrations of piracetam are able to improve mitochondrial dysfunction associated with oxidative stress and/or aging. Mitochondrial stabilization and protection might be an important mechanism to explain many of piracetam's beneficial effects in elderly patients. British Journal of Pharmacology (2006) 147, 199,208. doi:10.1038/sj.bjp.0706459 [source]