Dynamic Blood Concentrations of Aß1-40 and Aß1-42 in Alzheimer's Disease.

Amyloid-beta (Aß) is produced by the cleavage of amyloid precursor proteins in the cell membrane by ß-secretase and γ-secretase into a monomeric form with peptides of different lengths such as Aß1-40 or Aß1-42, which is then transformed into oligomeric and fibril forms and is considered to be one of the hallmarks of Alzheimer's disease (AD). The plasma concentrations of Aß1-40 and Aß1-42 are unstable after blood samples have been obtained. In order to examine the dynamic changes of plasma Aß1-42 and Aß1-40 in blood samples, we used fresh blood samples in ethylenediaminetetraacetic acid tubes from 32 clinically diagnosed AD patients. Each sample was subdivided into eight sub-samples, and levels of Aß1-40 and Aß1-42 were measured at 0 (baseline), 0.5, 1, 2, 3, 5, 8, and 24 h, respectively. All samples were incubated at 37°C before being measuring. The results showed that compared to baseline, 87.5 and 62.5% of the patients had higher plasma levels of Aß1-42 and Aß1-40 at 24 h, respectively. The patients with an increased amyloid level did not have a significantly different apo-lipoprotein E4 allele (APOE4) gene status for either Aß1-40 (p = 0.422) or Aß1-42 (p = 1.000). However, for plasma Aß1-42, the APOE4 carriers had a significantly lower level than the non-carriers at baseline [31.2 ± 6.5 (mean ± SD) ng/ml vs. 50.4 ± 47.7 ng/ml, p = 0.031] and 0.5 h (37.5 ± 7.6 ng/ml vs. 51.9 ± 30.8 ng/ml, p = 0.043). There were no significant differences between the APOE4 carriers and non-carriers in plasma Aß1-42 concentration at 1, 2, 3, 5, 8, and 24 h (p = 0.112, p = 0.086, p = 0.112, p = 0.263, p = 0.170 and p = 0.621, respectively). The Aß1-40 level was related to disease severity as assessed using the clinical dementia rating (CDR) scale. Patients with advanced stages of dementia (CDR = 1 and CDR = 2) had a significantly higher Aß1-40 level compared to those with very mild stage dementia (CDR = 0.5) at all time points (p < 0.05) except for 24 h (p = 0.059). Our findings illustrate the effects of APOE4 status on dynamic changes in plasma Aß1-40 and Aß1-42 levels, and significant associations between Aß1-40 level and disease severity. Further studies are needed to investigate the exact mechanisms of how APOE4 affects the dynamic changes in plasma Aß1-40 and Aß1-42, and the association between Aß1-40 and advanced dementia.

Activation of mitochondrial unfolded protein response is associated with Her2-overexpression breast cancer.

PURPOSE: Mitochondrial unfolding protein are abundant in breast cancer cells, but the mechanism by which breast cancer cells resist apoptosis is still not fully elucidated. In this study, we explored the role of mitochondrial unfolded protein response (mtUPR)-related proteins in four types of breast cancer tissues. METHODS: Mitochondrial fractions were taken from four breast cancer tissues (luminal A, luminal B, Her2 -overexpression, and TNBC) and the expression of mitochondrial polyubiquitinated proteins was observed by western blot and ELISA. In addition, the expression of hsp10, hsp60, and clpp in mitochondria was observed by western blot in breast cancer tissues and adjacent tissues, and confirmed by ELISA. The expression levels of hsp10 and hsp60 were correlated with clinicopathological parameters in 114 breast cancer patients. RESULTS: We found an increase in the performance of mitochondrial polyubiquitinated proteins in breast cancer tissues of luminal A, luminal B, Her2-overexpression, and TNBC. The mitochondrial hsp10, hsp60, and clpp are abundantly expressed in breast cancer tissues rather than adjacent noncancerous tissues. The expression levels of mitochondrial hsp10 and hsp60 were highest in histological grade 3 breast cancer tissues. Additionally, mitochondria with high hsp60 expression were more present in Her2-positive tumors. CONCLUSIONS: We observed that mtUPR was specifically activated in breast cancer tissues but inactivated in normal mammary tissue. MtUPR had also exhibited a particular increase in Her2-overexpression tumors but not in ER- or PR-positive tumors. Taken together, we suggested that mtUPR may act as a potential candidate for developing novel Her2-overexpression breast cancer therapy.

Impairment of mitochondrial unfolded protein response contribute to resistance declination of H2 O2 -induced injury in senescent MRC-5 cell model.

Accumulation of oxidative proteins within mitochondria leads to loss of mitochondrial function, which may lead to age-related degenerative diseases. Mitochondrial antioxidant defense capacity reflects the expression of mitochondrial unfolded protein response (mtUPR)-related proteins. Senescent cells are considered to be less resistant to cellular stress stimuli than exponentially growing cells. In this study, we aimed to investigate the ability of mitochondrial stress response in senescent cells to cope with the accumulation of mitochondrial unfolded proteins induced by hydrogen peroxide (H2 O2 ) and to understand the relevant molecular mechanisms. We report here that senescence-associated ß-galactosidase (SA-ß-gal) and senescence marker protein-30 (SMP-30), commonly used replicative senescence biomarkers, changed remarkably between population doubling (PD) 25 (exponentially growing cells) and PD50 (senescent cells) of MRC-5 fibroblasts. Mitochondrial unfolded proteins were significantly accumulated in H2 O2 -treated senescent cells, whereas mtUPR-related molecular chaperones (heat shock protein Hsp60 and Hsp10) and proteases (caseinolytic Clp protease) were not concomitantly elevated in senescent cells. In addition, decreased expression of stromal interacting molecule 1-Orai1-mediated store-operated Ca2+ entry following an declined intracellular calcium level after 2 mM calcium treatment together with H2 O2 addition, implying impairment of calcium influx in senescent MRC-5 during H2 O2 -induced injury. These findings suggest that senescent fibroblasts expressed higher vulnerability to H2 O2 -induced injury involving the imbalance of calcium homeostasis and impaired mitochondrial nuclear communication. This may provide useful information for the future development of therapeutic agents to prevent the adverse effects of aging on cells and the potential for treatment of proteinopathies in the elderly.

Differential Response of Non-cancerous and Malignant Breast Cancer Cells to Conditioned Medium of Adipose tissue-derived Stromal Cells (ASCs).

Background: The application of adipose tissue-derived stromal cells (ASCs) in regenerative medicine has become a growing trend due to its abundance and differentiation potentials. However, several breast cancer studies indicated that ASCs promote tumor progression, therefore, the use of ASCs for reconstruction after oncological surgery poses potential risks. In this study, we aimed to examine whether cancerous or non-cancerous breast cells will exhibit different responses to ASC-derived CM. Methods: ASCs were isolated from residuals of subcutaneous adipose tissue obtained from patients undergoing surgery. Cancerous MCF-7, MDA-MB231, and MDA-MB468 cell lines and one non-cancerous M10/H184B5F5 cell line were cultured with variant concentrations of ASC-derived conditioned medium (CM) for analysis. Results: ASC-derived CM significantly reduced cell viability by triggering apoptosis in MCF-7, MDA-MB231, and MDA-MB468 cell lines. ATM-Chk2-dependent DNA damage response was activated early in cancer cells when exposed to ASC-derived CM. By contrast, prompted cell proliferation instead of cell death was detected in M10/H184B5F5 cells under the treatment of lower CM concentration. Even when exposed to the highest concentration of CM, only cell cycle arrest accompanied by a weak DNA damage response were detected in M10/H184B5F5 cells, no cell deaths were observed. Conclusions: Overall, this study demonstrated that cancerous and non-cancerous breast cells respond differently to ASC-derived CM. ASC-derived CM triggered significant cell death in breast cancer cell lines, however non-cancerous breast cells exhibited dissimilar response to ASC-derived CM.

The risk of herpes simplex virus and human cytomegalovirus infection during pregnancy upon adverse pregnancy outcomes: A meta-analysis.

BACKGROUND AND OBJECTIVES: Herpes simplex virus (HSV) and human cytomegalovirus (HCMV) are widespread infections in humans, yet their impact on adverse pregnancy outcomes is controversial. The objective of this study was to evaluate the impact of HSV and HCMV infections during pregnancy on adverse pregnancy outcomes. METHODS: A systematic literature search was performed using Web of Science, Scopus, Medline, Embase, PubMed, and the Cochrane Library database for relevant publications up to 2nd August 2017. The odds ratio (OR) and relative risk (RR), and their corresponding 95% confidence intervals (CIs) were selected as the effect size. Statistical analysis was conducted using STATA 12.0. RESULTS: In total, 20 eligible studies were identified and included in the meta-analysis. Of these, 13 and 12 studies were related to the impact of HSV and HCMV upon adverse pregnancy outcomes, respectively. Collectively, the results indicated that HSV infection during pregnancy increased the risk of spontaneous abortion, premature birth and stillbirth with an OR of 3.81 (95% CI: 1.96-7.41), 3.83 (95% CI: 1.17-12.54), and 1.78 (95% CI: 1.08-2.95), respectively. HCMV infection during pregnancy also represented a risk factor for spontaneous abortion, premature birth and stillbirth with an OR of 1.61 (95% CI: 1.14-2.27), 1.86 (95% CI: 1.26-2.76) and 5.74 (95% CI: 2.04-16.12), respectively. CONCLUSIONS: Maternal HSV and HCMV infection during pregnancy increase the risk of spontaneous abortion, premature birth, and stillbirth.

Role of Exogenous Hsp72 on Liver Dysfunction during Sepsis.

This study examined the role of exogenous heat shock protein 72 (Hsp72) in reversing sepsis-induced liver dysfunction. Sepsis was induced by cecal ligation and puncture. Liver function was determined on the basis of the enzymatic activities of serum glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). Apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and caspase-9, and cleaved poly (ADP-ribose) polymerase (PARP) protein expressions were analyzed using Western blotting. Results showed GOT and GPT levels increased during sepsis, and levels were restored following the administration of human recombinant Hsp72 (rhHsp72). Increased liver tissue apoptosis was observed during sepsis, and normal apoptosis resumed on rhHsp72 administration. The Bcl-2/Bax ratio, cleaved caspase-3, caspase-9, and PARP protein expressions in the liver tissues were upregulated during sepsis and normalized after rhHsp72 treatment. We conclude that, during sepsis, exogenous Hsp72 restored liver dysfunction by inhibiting apoptosis via the mitochondria-initiated caspase pathway.

Nonlethal dose of silver nanoparticles attenuates TNF-α-induced hepatic epithelial cell death through HSP70 overexpression.

Silver nanoparticles (Ag-nps) have been widely used in various biomedical products. Compared with its hazardous effects extensively being studied, rare attention has been paid to the potential protective effect of Ag-nps to human health. The present study was designed to evaluate the protective effects of Ag-nps and heat shock treatment on tumor necrosis factor-α (TNF-α)-induced cell damage in Clone 9 cells. Clone 9 cells were pretreated with nonlethal concentration of Ag-nps (1 µg/ml) or heat shock, and then cell damages were induced by TNF-α (1 ng/ml). Protective effects of Ag-nps administration or heat shock treatment were determined by examining the TNF-α-induced changes in cell viabilities. The results showed that the intensity of cytotoxicity produced by TNF-α was alleviated upon treatment with nonlethal concentration of Ag-nps (1 µg/ml). Similar protective effects were also found upon heat shock treatment. These data demonstrate that Ag-nps and heat shock treatment were equally capable of inducing heat shock protein 70 (HSP70) protein expression in Clone 9 cells. The results suggest that clinically Ag-nps administration is a viable strategy to induce endogenous HSP70 expression instead of applying heat shock. In conclusion, our study for the first time provides evidence that Ag-nps may act as a viable alternative for HSP70 induction clinically.

Bioenergetic failure correlates with autophagy and apoptosis in rat liver following silver nanoparticle intraperitoneal administration.

BACKGROUND: Deposition and accumulation of silver nanoparticles (Ag-nps) in the liver have been shown to induce hepatotoxicity in animal studies. The hepatotoxicity may include oxidative stress, abnormalities in energy metabolism, and cell death. Studies have indicated that autophagy is an intracellular event involving balance of energy, nutrients, and turnover of subcellular organelles. The present study was undertaken to test the hypothesis that autophagy plays a role in mediating hepatotoxicity in animal after exposure to Ag-nps. Focus was placed on interrelationship between energy metabolism, autophagy, apoptosis and hepatic dysfunction. METHODS: Sprague Dawley rats were intraperitoneally injected with Ag-nps (10-30 nm in diameter) at concentration of 500 mg kg(-1). All animals were sacrificed on days 1, 4, 7, 10 and 30 after exposure and blood and liver tissues were collected for further studies. RESULTS: Uptake of Ag-nps was quite prompt and not proportional to the blood Ag concentration. Declination of ATP (-64% in days 1) and autophagy (determined by LC3-II protein expression and morphological evaluation) increased and peaked on the first day. The ATP content remained at low level even though the autophagy has been activated. Apoptosis (based on caspase-3 protein expression and TUNEL-positive cells staining) began to rise sigmoidally at days 1 and 4, reached a peak level at day 7, and remained at the same levels during days 7-30 post exposure. Meanwhile, autophagy exhibited a gradual decrease from days 1-10 and the decrease at day 30 was statistically significant as compared to day 0 (sham group). Inflammatory reaction (histopathological evaluation) was found at day 10 and preceded to an advanced degree at day 30 when liver function was impaired. CONCLUSIONS: These results indicate that following Ag-nps administration, autophagy was induced; however, failure to preserve autophagy compounded with energy reduction led to apoptosis and the eventual impairment of liver function. The study provides an in-vivo evidence of hepatotoxicity by continuous exposure of Ag-nps in rats.

Attenuation of mitochondrial unfolded protein response is associated with hepatic dysfunction in septic rats.

This study was conducted to reveal if the mitochondrial unfolded protein response (mtUPR), a conserved mitochondrial-nuclear communication mechanism, plays a critical role in the protein quality control system to cope with damaged protein during sepsis. Sepsis was induced by cecal ligation and puncture (CLP) in Sprague-Dawley rats. The efficiency of mtUPR was evaluated by measuring the transcriptional factors (CCAAT/enhancer-binder protein homologous protein [CHOP] and CCAAT/enhancer-binder protein-ß) and chaperones (heat shock protein 60 [Hsp60] and Hsp10) expression in response to hepatic mitochondrial oxidized proteins (carbonylated proteins, car-proteins) and multi-ubiquitinated proteins (ub-proteins). The results showed that car-proteins and ub-proteins were significantly increased at 9 and 18 h after CLP. In addition, serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase were significantly positively correlated with mitochondrial car-proteins and ub-proteins and negatively with intramitochondrial adenosine triphosphate. The expression of mitochondrial Hsp60 and Hsp10 decreased notably during the progression of sepsis, implying that failure of mtUPR occurred in the late septic liver. Interestingly, we evaluated the ratio of mitochondrial Hsp60/Hsp10 to the ub-proteins and found that both ratios were statistically lowered at the time points of 9 and 18 h in comparison with 3 and 6 h after CLP. These ratios were also significantly negatively correlated with glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase levels, suggesting that the ratios could act as an index of mtUPR failure and be a useful tool in estimating the ability of mitochondrial-nuclear communication in sepsis. In conclusion, the results indicated that mtUPR failure occurred during sepsis, and that the index of mtUPR may be a valuable measurement in assessing the severity of organ dysfunction in the clinical setting.

Inhibitory effect of hexahydrocurcumin on human platelet aggregation.

The effects of hexahydrocurcumin on adenosine diphosphate (ADP)-induced human platelet aggregation were studied. Treatment of human platelet-rich plasma with hexahydrocurcumin resulted in an inhibitory effect on platelet aggregation, suggesting the potential of this compound as an anti-atherosclerogenic agent in humans.

Hypoxia-inducible factor 1α regulates the expression of the mitochondrial ATPase inhibitor protein (IF1) in rat liver.

A growing number of reports indicate that bioenergetic failure plays a crucial role in the development of multiple organ failure during sepsis. Our previous results showed that the suppression of IF1 (mitochondrial ATPase inhibitor protein) expression and subsequent elevated mitochondrial F(o)F1-ATPase activity might contribute to the bioenergetic failure in the liver during sepsis, and the influence of the decreased transcriptional level of IF1 might be an important factor. In this study, we investigated the interaction of IF1 protein expression and hypoxia-inducible factor 1 (HIF-1), a transcription factor that is correlated with the inflammatory status in sepsis. The results showed that nuclear HIF-1α protein, a subunit of HIF-1, and IF1 mRNA expression were coincidently reduced in late septic liver of rats. Furthermore, in vitro, overexpression of HIF-1α by hypoxia or CoCl2 (HIF-1α activator) treatment augmented IF1 protein levels. On the contrary, HIF-1α antisense oligonucleotide and siRNA were used to specifically downregulate HIF-1α expression, and then IF1 protein levels were significantly decreased in clone 9 cells. Meanwhile, downregulation of HIF-1α expression led to elevate the mitochondrial F(o)F1-ATPase activity in the presence of Bis-Tris buffer (pH 6.5). In conclusion, these results suggested for the first time that the HIF-1 might play a crucial role in regulating IF1 protein expression in late septic liver.

Lipopolysaccharide-stimulated leukocytes contribute to platelet aggregative dysfunction, which is attenuated by catalase in rats.

Endotoxemia causes several hematological dysfunctions, including platelet degranulation or disseminated intravascular coagulation, which lead to thrombotic and hemorrhagic events. Here, we tested the hypothesis that bacterial lipopolysaccharide (LPS)-stimulated leukocytes contribute to platelet aggregative dysfunction, and this function is attenuated by antioxidants. Platelet-rich plasma (PRP) was prepared from whole blood of normal and endotoxemic rats. The ability of platelet aggregation was measured by an aggregometer. LPS (50-100 µg/mL) was incubated with PRP, whole blood and PRP with polymorphonuclear leukocytes (PMNs) for 30 minutes, 60 minutes and 90 minutes, and platelet aggregation was detected. LPS-induced platelet aggregative dysfunction was undetectable in intact PRP which was isolated from normal whole blood, whereas it was detected in PRP isolated from endotoxemic rats and LPS-treated whole blood. Moreover, the effect of LPS-induced platelet aggregative dysfunction on intact PRP was observed when the PMNs were added. LPS-induced platelet aggregative dysfunction was significantly attenuated by catalase alone and in combination with N(G)-nitro-L-arginine methyl ester, but not by N(G)-nitro-L-arginine methyl ester alone. These results indicate that LPS-stimulated PMNs modulate platelet aggregation during LPS treatment and the effects are reversed by antioxidants. PMNs serve as an approach to understand LPS-induced platelet aggregative dysfunction during endotoxemia. During this process, the generation of reactive oxygen species, hydrogen peroxide especially, from LPS-stimulated PMNs could be an important potential factor in LPS-induced platelet aggregative dysfunction. Catalase contributes to the prevention of platelet dysfunction during LPS-induced sepsis.

Plethysmographic lung volumes in children with sighing dyspnea.

BACKGROUND: This study compared the plethysmographic lung volumes of children with sighing dyspnea with healthy children and tested the hypothesis that sighing children suffer from hyperinflation or gas trapping as a cause of dyspnea. METHODS: From January 2006 to December 2006, pediatric patients with sighing dyspnea presenting to the pulmonary clinic of a tertiary children's hospital who had no apparent cardiopulmonary diseases were prospectively enrolled; normal healthy children were invited to participate for comparison. Baseline pre-bronchodilator spirometry and post-inhaled bronchodilator spirometry were measured for the determination of bronchodilator response. Plethysmographic lung volumes were determined solely for total lung capacity, residual volume (RV) and functional residual capacity (FRC) without the use of inhaled bronchodilator according to standard procedure. RESULTS: Eighteen sighing children (10 boys) and 10 healthy subjects (six boys) were included in the present study. They had a median age of 13 years (range, 8-15 years) and 13 years (range, 8-17 years), respectively. The mean baseline forced vital capacity (FVC) of subjects with dyspnea was 79.4 +/- 16.7% of predicted, while that of the normal control children was 88.4 +/- 6.7%, which was not statistically significantly different. Forced expiratory volume in 1 s (FEV(1)), FEV(1)/FVC % of predicted were within normal limits and indicated no bronchodilator response. RV and RV/total lung capacity (TLC) were elevated in children with sighing dyspnea that were not measured by spirometry, but TLC and FRC measured on plethysmography (FRC(pleth)) were not increased. CONCLUSIONS: RV and RV/TLC were higher in children with sighing dyspnea that were not measured by spirometry, but TLC and FRC(pleth) were not increased. The causal link between dysfunctional breathing patterns and changes in static lung volumes was not able to be determined in the present study. The possibility of heterogeneity of patients with sighing dyspnea obscures the significance of lung volume discrepancy in this population; further subdivision of children with sighing dyspnea in a larger cohort of patients is required.

Solvent effects on extraction and HPLC analysis of soybean isoflavones and variations of isoflavone compositions as affected by crop season.

Spring (February to June) and fall (August to December) crops of soybean grown yearly in Taiwan with reverse temperature patterns provide a novel model to assess the effect of the crop season. In this study, three soybean cultivars, namely CH 1, VS-KS 2, and HBS, were grown for 2001 fall, 2002 spring, 2003 fall, 2004 spring, 2004 fall, and 2005 spring crops. The harvested and sun-dried soybeans were lyophilized, pulverized, and stored at -25 degrees C until HPLC analyses of isoflavone compositions were performed. As affected by extraction solvent and HPLC mobile phase, the amount of isoflavones extracted by methanol-H(2)O was higher than those extracted by acetic acid-acetonitrile. In addition, when both extracts were subjected to HPLC analysis with reversed C18 column run respectively with methanol-H(2)O and acetic acid-acetonitrile mobile phases, malonyldaidzin, malonylglycitin, and malonylgenistin were not detected in the former phase. Accordingly, all harvested soybeans were subjected to methanol-H(2)O extraction and HPLC analysis with the acetic acid-acetonitrile mobile phase. Among the detected soybeans, daidzin, genistin, malonyldaidzin, and malonylgenistin were the majors and glycitin, malonylglycitin, daidzein, and genistein were the minors of isoflavones. As affected by crop season for each cultivar grown for 3 years, daidzin, genistin, malonyldaidzin, and malonylgenistin contents of soybeans of the fall crops were significantly higher than those of their spring crops ( p < 0.05).

Suppression of mitochondrial ATPase inhibitor protein (IF1) in the liver of late septic rats.

Sepsis and ensuing multiple organ failure continue to be the most leading cause of death in critically ill patients. Despite hepatocyte-related dysfunctions such as necrosis, apoptosis as well as mitochondrial damage are observed in the process of sepsis, the molecular mechanism of pathogenesis remains uncertain. We recently identified one of the differentially expressed genes, mitochondrial ATPase inhibitor protein (IF1) which is down-regulated in late septic liver. Hence, we further hypothesized that the variation of IF1 protein may be one of the causal events of the hepatic dysfunction during late sepsis. The results showed that the elevated mitochondrial F0F1-ATPase activity is concomitant with the decline of intramitochondrial ATP concentration in late septic liver. In addition, the key finding of this study showed that the mRNA and the mitochondrial content of IF1 were decreased in late sepsis while no detectable IF1 was found in cytoplasm. When analyzed by immunoprecipitation, it seems reasonable to imply that the association capability of IF1 with F1-ATPase beta-subunit is not affected. These results confirm the first evidence showing that the suppression of IF1 expression and subsequent elevated mitochondrial F0F1-ATPase activity might contribute to the bioenergetic failure in the liver during late sepsis.

Role of cAMP-response element-binding protein phosphorylation in hepatic apoptosis under protein kinase C alpha suppression during sepsis.

Previous studies have shown that a decrease in protein kinase C (PKC) alpha levels contributes to hepatic failure and/or apoptosis during sepsis, and suppression of PKCalpha plays a critical role in triggering caspase-dependent apoptosis, which can modulate expression of Bcl-xL. However, the underlying molecular mechanism remains uncertain. In the present study, we examined whether a decrease in the nuclear PKCalpha levels causes hepatic apoptosis via modulation of cAMP-response element-binding protein (CREB) or nuclear factor-kappaB (NFkappaB), the crucial factors regulating the expression of prosurvival Bcl-xL. For polymicrobial sepsis induction, a cecal ligation and puncture model was used; at 9 or 18 h after CLP, experiments were terminated, referring as early or late sepsis, respectively. Additionally, PKCalpha was suppressed by stable transfection of antisense PKCalpha plasmid into a Clone-9 rat hepatic epithelial cell. The results showed that the nuclear PKCalpha was significantly decreased in the liver during sepsis, which was accompanied by decreases in phospho-CREB content, DNA-binding activity of CREB, and Bcl-xL expression. Likewise, the binding activity of NFkappaB increased significantly, which was associated with a decrease in cytosolic inhibitory-kappaBalpha content. The in vitro suppression of PKCalpha also resulted in decreases in the phospho-CREB content and DNA-binding activity, which were accompanied by down-regulation of Bcl-xL and apoptosis, but no significant alteration in NFkappaB-binding activity. The in vivo and in vitro results suggest that the suppression of PKCalpha results in a decreased CREB phosphorylation and subsequent down-regulation of Bcl-xL, which may contribute to the hepatic apoptosis during sepsis.

The essential role of PKCalpha in the protective effect of heat-shock pretreatment on TNFalpha-induced apoptosis in hepatic epithelial cell line.

During sepsis, hepatic apoptosis occurred, which is associated with inactivation of PKCalpha and elevation of tumor necrosis factor-alpha (TNFalpha), an apoptosis trigger. Heat shock, accompanied by the increase of heat-shock protein (Hsp72), has been shown to exhibit a protective role on cell survival. However, Hsp72 was unable to express during sepsis when the apoptosis was markedly increased. We hypothesized that hepatic apoptosis during sepsis may be due to the failure to induce expression of Hsp72, which is activated by PKC-phosphorylated HSF. This study was designed to examine the role of PKCalpha in Hsp72 expression and the anti-apoptotic effect of Hsp72 on hepatic epithelial cells by analyzing a TNFalpha-induced apoptosis system. The following results were observed: (1) Hsp72 was highly expressed at 8 h after heat-shock treatment in a clone 9 hepatic epithelial cell line; (2) the protein expression of PKCalpha in membrane-associated fraction was decreased by TNFalpha treatment; (3) the TNFalpha-induced cell death, especially apoptosis, was diminished by heat-shock pretreatment; (4) in the presence of PKCalpha antisense, which blocks the PKCalpha resynthesis, no protective effect of heat-shock pretreatment was observed, and the protein expression of Hsp72 was significantly suppressed. These results suggest that PKCalpha plays a critical role in the expression of Hsp72, which subsequently protects against TNFalpha-induced hepatic apoptosis.