Effect of substituents at the C3´, C3´N, C10 and C2-meta-benzoate positions of taxane derivatives on their activity against resistant cancer cells.

We tested the effect of substituents at the (1) C3´, C3´N, (2) C10, and (3) C2-meta-benzoate positions of taxane derivatives on their activity against sensitive versus counterpart paclitaxel-resistant breast (MCF-7) and ovarian (SK-OV-3) cancer cells. We found that (1) non-aromatic groups at both C3´ and C3´N positions, when compared with phenyl groups at the same positions of a taxane derivative, significantly reduced the resistance of ABCB1 expressing MCF-7/PacR and SK-OV-3/PacR cancer cells. This is, at least in the case of the SB-T-1216 series, accompanied by an ineffective decrease of intracellular levels in MCF-7/PacR cells. The low binding affinity of SB-T-1216 in the ABCB1 binding cavity can elucidate these effects. (2) Cyclopropanecarbonyl group at the C10 position, when compared with the H atom, seems to increase the potency and capability of the derivative in overcoming paclitaxel resistance in both models. (3) Derivatives with fluorine and methyl substituents at the C2-meta-benzoate position were variously potent against sensitive and resistant cancer cells. All C2 derivatives were less capable of overcoming acquired resistance to paclitaxel in vitro than non-substituted analogs. Notably, fluorine derivatives SB-T-121205 and 121,206 were more potent against sensitive and resistant SK-OV-3 cells, and derivatives SB-T-121405 and 121,406 were more potent against sensitive and resistant MCF-7 cells. (4) The various structure-activity relationships of SB-T derivatives observed in two cell line models known to express ABCB1 favor their complex interaction not based solely on ABCB1.

Expression profiles of iron transport molecules along the duodenum.

Duodenal biopsies are considered a suitable source of enterocytes for studies of dietary iron absorption. However, the expression level of molecules involved in iron absorption may vary along the length of duodenum. We aimed to determine whether the expression of molecules involved in the absorption of heme and non-heme iron differs depending on the location in the duodenum. Analysis was performed with samples of duodenal biopsies from 10 individuals with normal iron metabolism. Samples were collected at the following locations: (a) immediately post-bulbar, (b) 1-2 cm below the papilla of Vater and (c) in the distal duodenum. The gene expression was analyzed at the mRNA and protein level using real-time PCR and Western blot analysis. At the mRNA level, significantly different expression of HCP1, DMT1, ferroportin and Zip8 was found at individual positions of duodenum. Position-dependent expression of other molecules, especially of FLVCR1, HMOX1 and HMOX2 was also detected but with no statistical significances. At the protein level, we observed statistically significantly decreasing expression of transporters HCP1, FLVCR1, DMT1, ferroportin, Zip14 and Zip8 with advancing positions of duodenum. Our results are consistent with a gradient of diminishing iron absorption along the duodenum for both heme and non-heme iron.

Molecular Mechanisms of Apoptosis Induction and Its Regulation by Fatty Acids in Pancreatic ß-Cells.

Pancreatic ß-cell failure and death contribute significantly to the pathogenesis of type 2 diabetes. One of the main factors responsible for ß-cell dysfunction and subsequent cell death is chronic exposure to increased concentrations of FAs (fatty acids). The effect of FAs seems to depend particularly on the degree of their saturation. Saturated FAs induce apoptosis in pancreatic ß-cells, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction by saturated FAs in ß-cells are not completely elucidated. Saturated FAs induce ER stress, which in turn leads to activation of all ER stress pathways. When ER stress is severe or prolonged, apoptosis is induced. The main mediator seems to be the CHOP transcription factor. Via regulation of expression/activity of pro- and anti-apoptotic Bcl-2 family members, and potentially also through the increase in ROS production, CHOP switches on the mitochondrial pathway of apoptosis induction. ER stress signalling also possibly leads to autophagy signalling, which may activate caspase-8. Saturated FAs activate or inhibit various signalling pathways, i.e., p38 MAPK signalling, ERK signalling, ceramide signalling, Akt signalling and PKCδ signalling. This may lead to the activation of the mitochondrial pathway of apoptosis, as well. Particularly, the inhibition of the pro-survival Akt signalling seems to play an important role. This inhibition may be mediated by multiple pathways (e.g., ER stress signalling, PKCδ and ceramide) and could also consequence in autophagy signalling. Experimental evidence indicates the involvement of certain miRNAs in mechanisms of FA-induced ß-cell apoptosis, as well. In the rather rare situations when unsaturated FAs are also shown to be pro-apoptotic, the mechanisms mediating this effect in ß-cells seem to be the same as for saturated FAs. To conclude, FA-induced apoptosis rather appears to be preceded by complex cross talks of multiple signalling pathways. Some of these pathways may be regulated by decreased membrane fluidity due to saturated FA incorporation. Few data are available concerning molecular mechanisms mediating the protective effect of unsaturated FAs on the effect of saturated FAs. It seems that the main possible mechanism represents a rather inhibitory intervention into saturated FA-induced pro-apoptotic signalling than activation of some pro-survival signalling pathway(s) or metabolic interference in ß-cells. This inhibitory intervention may be due to an increase of membrane fluidity.

The Role of TRIP6, ABCC3 and CPS1 Expression in Resistance of Ovarian Cancer to Taxanes.

The main problem precluding successful therapy with conventional taxanes is de novo or acquired resistance to taxanes. Therefore, novel experimental taxane derivatives (Stony Brook taxanes; SB-Ts) are synthesized and tested as potential drugs against resistant solid tumors. Recently, we reported alterations in ABCC3, CPS1, and TRIP6 gene expression in a breast cancer cell line resistant to paclitaxel. The present study aimed to investigate gene expression changes of these three candidate molecules in the highly resistant ovarian carcinoma cells in vitro and corresponding in vivo models treated with paclitaxel and new experimental Stony Brook taxanes of the third generation (SB-T-121605 and SB-T-121606). We also addressed their prognostic meaning in ovarian carcinoma patients treated with taxanes. We estimated and observed changes in mRNA and protein profiles of ABCC3, CPS1, and TRIP6 in resistant and sensitive ovarian cancer cells and after the treatment of resistant ovarian cancer models with paclitaxel and Stony Brook taxanes in vitro and in vivo. Combining Stony Brook taxanes with paclitaxel caused downregulation of CPS1 in the paclitaxel-resistant mouse xenograft tumor model in vivo. Moreover, CPS1 overexpression seems to play a role of a prognostic biomarker of epithelial ovarian carcinoma patients' poor survival. ABCC3 was overexpressed in EOC tumors, but after the treatment with taxanes, its up-regulation disappeared. Based on our results, we can suggest ABCC3 and CPS1 for further investigations as potential therapeutic targets in human cancers.

Origin of the 31 P MR signal at 5.3 ppm in patients with critical limb ischemia.

An unknown intense signal (Pun ) with a mean chemical shift of 5.3 ppm was observed in 31 P MR spectra from the calf muscles of patients with the diabetic foot syndrome. The aim of the study was to identify the origin of this signal and its potential as a biomarker of muscle injury. Calf muscles of 68 diabetic patients (66.3 ± 8.6 years; body mass index = 28.2 ± 4.3 kg/m2 ) and 12 age-matched healthy controls were examined by (dynamic) 31 P MRS (3 T system, 31 P/1 H coil). Phantoms (glucose-1-phosphate, Pi and PCr) were measured at pH values of 7.05 and 7.51. At rest, Pun signals with intensities higher than 50% of the Pi intensity were observed in 10 of the 68 examined diabetic subjects. We tested two hypothetical origins of the Pun signal: (1) phosphorus from phosphoesters and (2) phosphorus from extra- and intracellular alkaline phosphate pools. 2,3-diphosphoglycerate and glucose-1-phosphate are the only phosphoesters with signals in the chemical shift region close to 5.3 ppm. Both compounds can be excluded: 2,3-diphosphoglycerate due to the missing second signal component at 6.31 ppm; glucose-1-phosphate because its chemical shifts are about 0.2 ppm downfield from the Pi signal (4.9 ppm). If the Pun signal is from phosphate, it represents a pH value of 7.54 ± 0.05. Therefore, it could correspond to signals of Pi in mitochondria. However, patients with critical limb ischemia have rather few mitochondria and so the Pun signal probably originates from interstitia. Our data suggest that the increased Pun signal observed in patients with the diabetic foot syndrome is a biomarker of severe muscular damage.

Hypertriglyceridemia and atherosclerosis risk.

The history of studying hypertriglyceridemia as a risk factor for atherosclerosis has been going on for a half a century. The significance of this parameter as measured in fasting state is not entirely clear, since the statistical significance between triglyceride concentration and cardiovascular risk is lost after adjustment to HDL-cholesterol concentration. Remnant particles of chylomicrons and very low density lipoproteins measured postprandially appear to be responsible for the risk associated with hypertriglyceridemia. As the concentration of non-fasting triglycerides increases, the risk of myocardial infarction increases gradually up to five times.

Hypoxia Modulates Effects of Fatty Acids on NES2Y Human Pancreatic ß-cells.

Saturated fatty acids (FAs) induce apoptosis in the human pancreatic NES2Y ß-cell line while unsaturated FAs have nearly no detrimental effect. Moreover, unsaturated FAs are capable of inhibiting the pro-apoptotic effect of saturated FAs. Hypoxia is also known to have deleterious effects on ß-cells function and viability. In the present study, we have tested the modulatory effect of hypoxia on the effect of FAs on the growth and viability of the human pancreatic NES2Y ß-cells. This study represents the first study testing hypoxia effect on effects of FAs in pancreatic ß-cells as well as in other cell types. We showed that hypoxia increased the pro-apoptotic effect of saturated stearic acid (SA). Endoplasmic reticulum stress signaling seemed to be involved while redistribution of FA transporters fatty acid translocase/cluster of differentiation 36 (FAT/CD36) and fatty acid-binding protein (FABP) do not seem to be involved in this effect. Hypoxia also strongly decreased the protective effect of unsaturated oleic acid (OA) against the pro-apoptotic effect of SA. Thus, in the presence of hypoxia, OA was unable to save SA-treated ß-cells from apoptosis induction. Hypoxia itself had only a weak detrimental effect on NES2Y cells. Our data suggest that hypoxia could represent an important factor in pancreatic ß-cell death induced and regulated by FAs and thus in the development of type 2 diabetes mellitus.

Differentially Expressed Mitochondrial Proteins in Human MCF7 Breast Cancer Cells Resistant to Paclitaxel.

Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.

Substituents at the C3' and C3'N positions are critical for taxanes to overcome acquired resistance of cancer cells to paclitaxel.

We tested the role of substituents at the C3' and C3'N positions of the taxane molecule to identify taxane derivatives capable of overcoming acquired resistance to paclitaxel. Paclitaxel-resistant sublines SK-BR-3/PacR and MCF-7/PacR as well as the original paclitaxel-sensitive breast cancer cell lines SK-BR-3 and MCF-7 were used for testing. Increased expression of the ABCB1 transporter was found to be involved in the acquired resistance. We tested three groups of taxane derivatives: (1) phenyl group at both C3' and C3'N positions, (2) one phenyl at one of the C3' and C3'N positions and a non-aromatic group at the second position, (3) a non-aromatic group at both C3' and C3'N positions. We found that the presence of phenyl groups at both C3' and C3'N positions is associated with low capability of overcoming acquired paclitaxel resistance compared to taxanes containing at least one non-aromatic substituent at the C3' and C3'N positions. The increase in the ATPase activity of ABCB1 transporter after the application of taxanes from the first group was found to be somewhat higher than after the application of taxanes from the third group. Molecular docking studies demonstrated that the docking score was the lowest, i.e. the highest binding affinity, for taxanes from the first group. It was intermediate for taxanes from the second group, and the highest for taxanes from the third group. We conclude that at least one non-aromatic group at the C3' and C3'N positions of the taxane structure, resulting in reduced affinity to the ABCB1 transporter, brings about high capability of taxane to overcome acquired resistance of breast cancer cells to paclitaxel, due to less efficient transport of the taxane compound out of the cancer cells.

Transcript expression and genetic variability analysis of caspases in breast carcinomas suggests CASP9 as the most interesting target.

BACKGROUND: Apoptosis plays a critical role in cancer cell survival and tumor development. We provide a hypothesis-generating screen for further research by exploring the expression profile and genetic variability of caspases (2, 3, 7, 8, 9, and 10) in breast carcinoma patients. This study addressed isoform-specific caspase transcript expression and genetic variability in regulatory sequences of caspases 2 and 9. METHODS: Gene expression profiling was performed by quantitative real-time PCR in tumor and paired non-malignant tissues of two independent groups of patients. Genetic variability was determined by high resolution melting, allelic discrimination, and sequencing analysis in tumor and peripheral blood lymphocyte DNA of the patients. RESULTS: CASP3 A+B and S isoforms were over-expressed in tumors of both patient groups. The CASP9 transcript was down-regulated in tumors of both groups of patients and significantly associated with expression of hormonal receptors and with the presence of rs4645978-rs2020903-rs4646034 haplotype in the CASP9 gene. Patients with a low intratumoral CASP9A/B isoform expression ratio (predicted to shift equilibrium towards anti-apoptotic isoform) subsequently treated with adjuvant chemotherapy had a significantly shorter disease-free survival than those with the high ratio (p=0.04). Inheritance of CC genotype of rs2020903 in CASP9 was associated with progesterone receptor expression in tumors (p=0.003). CONCLUSIONS: Genetic variability in CASP9 and expression of its splicing variants present targets for further study.

Effect of Saturated Stearic Acid on MAP Kinase and ER Stress Signaling Pathways during Apoptosis Induction in Human Pancreatic ß-Cells Is Inhibited by Unsaturated Oleic Acid.

It has been shown that saturated fatty acids (FAs) have a detrimental effect on pancreatic ß-cells function and survival, leading to apoptosis, whereas unsaturated FAs are well tolerated and are even capable of inhibiting the pro-apoptotic effect of saturated FAs. Molecular mechanisms of apoptosis induction and regulation by FAs in ß-cells remain unclear; however, mitogen-activated protein (MAP) kinase and endoplasmic reticulum (ER) stress signaling pathways may be involved. In this study, we tested how unsaturated oleic acid (OA) affects the effect of saturated stearic acid (SA) on the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways as well as the ER stress signaling pathways during apoptosis induction in the human pancreatic ß-cells NES2Y. We demonstrated that OA is able to inhibit all effects of SA. OA alone has only minimal or no effects on tested signaling in NES2Y cells. The point of OA inhibitory intervention in SA-induced apoptotic signaling thus seems to be located upstream of the discussed signaling pathways.

Characterization of acquired paclitaxel resistance of breast cancer cells and involvement of ABC transporters.

Development of taxane resistance has become clinically very important issue. The molecular mechanisms underlying the resistance are still unclear. To address this issue, we established paclitaxel-resistant sublines of the SK-BR-3 and MCF-7 breast cancer cell lines that are capable of long-term proliferation in 100nM and 300nM paclitaxel, respectively. Application of these concentrations leads to cell death in the original counterpart cells. Both sublines are cross-resistant to doxorubicin, indicating the presence of the MDR phenotype. Interestingly, resistance in both paclitaxel-resistant sublines is circumvented by the second-generation taxane SB-T-1216. Moreover, we demonstrated that it was not possible to establish sublines of SK-BR-3 and MCF-7 cells resistant to this taxane. It means that at least the tested breast cancer cells are unable to develop resistance to some taxanes. Employing mRNA expression profiling of all known human ABC transporters and subsequent Western blot analysis of the expression of selected transporters, we demonstrated that only the ABCB1/PgP and ABCC3/MRP3 proteins were up-regulated in both paclitaxel-resistant sublines. We found up-regulation of ABCG2/BCRP and ABCC4 proteins only in paclitaxel-resistant SK-BR-3 cells. In paclitaxel-resistant MCF-7 cells, ABCB4/MDR3 and ABCC2/MRP2 proteins were up-regulated. Silencing of ABCB1 expression using specific siRNA increased significantly, but did not completely restore full sensitivity to both paclitaxel and doxorubicin. Thus we showed a key, but not exclusive, role for ABCB1 in mechanisms of paclitaxel resistance. It suggests the involvement of multiple mechanisms in paclitaxel resistance in tested breast cancer cells.

Differentially expressed proteins in human MCF-7 breast cancer cells sensitive and resistant to paclitaxel.

Resistance of cancer cells to chemotherapeutic agents is one of the main causes of treatment failure. In order to detect proteins potentially involved in the mechanism of resistance to taxanes, we assessed differences in protein expression in MCF-7 breast cancer cells that are sensitive to paclitaxel and in the same cells with acquired resistance to paclitaxel (established in our lab). Proteins were separated using two-dimensional electrophoresis. Changes in their expression were determined and proteins with altered expression were identified using mass spectrometry. Changes in their expression were confirmed using western blot analysis. With these techniques, we found three proteins expressed differently in resistant MCF-7 cells, i.e., thyroid hormone-interacting protein 6 (TRIP6; upregulated to 650%), heat shock protein 27 (HSP27; downregulated to 50%) and cathepsin D (downregulated to 28%). Silencing of TRIP6 expression by specific siRNA leads to decreased number of grown resistant MCF-7 cells. In the present study we have pointed at some new directions in the studies of the mechanism of resistance to paclitaxel in breast cancer cells.

Diurnal variation in cholesterol 7α-hydroxylase activity is determined by the -203A>C polymorphism of the CYP7A1 gene.

AIM: To determine whether the promoter polymorphism -203A>C of cholesterol-7α-hydroxylase encoding gene (CYP7A1) affects diurnal variation in CYP7A1 enzyme activity. METHODS: The study included 16 healthy male volunteers - 8 homozygous for -203A and 8 homozygous for the -203C allele of CYP7A1. Three 15-hour examinations (from 7am to 10pm) were carried out for each of the participants: after one-day treatment with cholestyramine; after one-day treatment with chenodeoxycholic acid (CDCA); and a control examination without any treatment. The plasma concentration of 7α-hydroxy-4-cholesten-3-one (C4), a marker of CYP7A1 activity, was determined in all the experiments at 90-min intervals. RESULTS: CYP7A1 activity was up-regulated after treatment with cholestyramine and suppressed after treatment with CDCA. There were no differences between -203A and -203C allele carriers in the response of enzyme activity to both drugs. In the control experiment, -203A allele carriers displayed diurnal variation in enzyme activity, whereas CYP7A1 activity did not change in -203C allele carriers. These results were confirmed by modeling the dynamics of C4 using polynomial regression. CONCLUSION: The promoter polymorphism of the CYP7A1 gene has a pronounced impact on diurnal variation in CYP7A1 activity.

Kinase Signaling in Apoptosis Induced by Saturated Fatty Acids in Pancreatic ß-Cells.

Pancreatic ß-cell failure and death is considered to be one of the main factors responsible for type 2 diabetes. It is caused by, in addition to hyperglycemia, chronic exposure to increased concentrations of fatty acids, mainly saturated fatty acids. Molecular mechanisms of apoptosis induction by saturated fatty acids in ß-cells are not completely clear. It has been proposed that kinase signaling could be involved, particularly, c-Jun N-terminal kinase (JNK), protein kinase C (PKC), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt kinases and their pathways. In this review, we discuss these kinases and their signaling pathways with respect to their possible role in apoptosis induction by saturated fatty acids in pancreatic ß-cells.

p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic ß-Cells.

Saturated stearic acid (SA) induces apoptosis in the human pancreatic ß-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic ß-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis.

The role of individual caspases in cell death induction by taxanes in breast cancer cells.

BACKGROUND: In previous study we showed that caspase-2 plays the role of an apical caspase in cell death induction by taxanes in breast cancer cells. This study deals with the role of other caspases. We tested breast cancer cell lines SK-BR-3 (functional caspase-3) and MCF-7 (nonfunctional caspase-3). METHODS AND RESULTS: Using western blot analysis we demonstrated the activation of initiator caspase-8 and -9 as well as executioner caspase-6 and -7 in both tested cell lines after application of taxanes (paclitaxel, SB-T-1216) at death-inducing concentrations. Caspase-3 activation was also found in SK-BR-3 cells. Employing specific siRNAs after taxane application, suppression of caspase-3 expression significantly increased the number of surviving SK-BR-3 cells. Inhibition of caspase-7 expression also increased the number of surviving SK-BR-3 and MCF-7 cells. On the other hand, suppression of caspase-8 and caspase-9 expression had no significant effect on cell survival. However, caspase-9 seemed to be involved in the activation of caspase-3 and caspase-7. Caspase-3 and caspase-7 appeared to activate mutually. Furthermore, we observed a significant decrease in mitochondrial membrane potential (flow cytometric analysis) and cytochrome c release (confocal microscopy, western blot after cell fractionation) from mitochondria in SK-BR-3 cells. No such changes were observed in MCF-7 cells after taxane treatment. CONCLUSION: We conclude that the activation of apical caspase-2 results in the activation of caspase-3 and -7 without the involvement of mitochondria. Caspase-9 can be activated directly via caspase-2 or alternatively after cytochrome c release from mitochondria. Subsequently, caspase-9 activation can also lead to caspase-3 and -7 activations. Caspase-3 and caspase-7 activate mutually. It seems that there is also a parallel pathway involving mitochondria that can cooperate in taxane-induced cell death in breast cancer cells.

Mitochondrial function in skeletal muscle of patients with protracted critical illness and ICU-acquired weakness.

BACKGROUND: Mitochondrial damage occurs in the acute phase of critical illness, followed by activation of mitochondrial biogenesis in survivors. It has been hypothesized that bioenergetics failure of skeletal muscle may contribute to the development of ICU-acquired weakness. The aim of the present study was to determine whether mitochondrial dysfunction persists until protracted phase of critical illness. METHODS: In this single-centre controlled-cohort ex vivo proof-of-concept pilot study, we obtained vastus lateralis biopsies from ventilated patients with ICU-acquired weakness (n = 8) and from age and sex-matched metabolically healthy controls (n = 8). Mitochondrial functional indices were measured in cytosolic context by high-resolution respirometry in tissue homogenates, activities of respiratory complexes by spectrophotometry and individual functional capacities were correlated with concentrations of electron transport chain key subunits from respiratory complexes II, III, IV and V measured by western blot. RESULTS: The ability of aerobic ATP synthesis (OXPHOS) was reduced to ~54% in ICU patients (p<0.01), in correlation with the depletion of complexes III (~38% of control, p = 0.02) and IV (~26% of controls, p<0.01) and without signs of mitochondrial uncoupling. When mitochondrial functional indices were adjusted to citrate synthase activity, OXPHOS and the activity of complexes I and IV were not different, whilst the activities of complexes II and III were increased in ICU patients 3-fold (p<0.01) respectively 2-fold (p<0.01). CONCLUSIONS: Compared to healthy controls, in ICU patients we have demonstrated a ~50% reduction of the ability of skeletal muscle to synthetize ATP in mitochondria. We found a depletion of complex III and IV concentrations and relative increases in functional capacities of complex II and glycerol-3-phosphate dehydrogenase/complex III.

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 µM, 1 µM, 10 µM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 µM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 µM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE.

The effect of cultureware surfaces on functional and structural components of differentiated 3T3-L1 preadipocytes.

Experiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.