Microlearning activities improve student comprehension of difficult concepts and performance in a biochemistry course.

INTRODUCTION: Students must rapidly learn and retain fundamental basic science knowledge in a doctor of pharmacy curriculum. Active learning stimulates engagement, reinforces concept understanding, and promotes retention of knowledge. The purpose this study was to determine if introducing game-based active recall and critical thinking microlearning activities improved student comprehension of challenging concepts, exam performance, and successful completion of a biochemistry course. METHODS: Microlearning activities were generated using Articulate Storyline software. Questions and problems were embedded in gamification-type activities to reinforce challenging biochemistry concepts and improve critical thinking. Activities were published on Blackboard and student performance was recorded. Students were divided into performance groups using their first exam scores. Student's exam scores were related to results from corresponding microlearning. Statistical analysis of results was performed to compare exam results with outcomes of microlearning activities. RESULTS: Student performance on exams and final scores positively correlated with successful completion of microlearning activities. Students who successfully completed more microlearning activities performed significantly better on all exams in comparison with students who completed few microlearning activities. Students who initially struggled with the material but completed more microlearning improved their performance on exams and passed the course with a higher score. In contrast, students who struggled and completed fewer activities failed to improve their exam and course performance. CONCLUSIONS: Active recall and critical thinking microlearning activities enhanced knowledge retention and comprehension of challenging biochemical concepts. Microlearning scores positively correlated with student exam performance in a biochemistry course, especially amongst students struggling with the material.

Thin-cap fibroatheroma and increased coronary intima-media thickness are associated with an altered balance of arginine metabolites.

INTRODUCTION: Arginase inhibition increases plasma citrulline and citrulline / ornithine (C/O) ratio, and reduces plasma ornithine and ornithine / arginine (O/A) ratio in an animal model of myocardial infarction (MI). OBJECTIVES: We hypothesized that the presence of thin­cap fibroatheroma (TCFA) in the culprit lesion and increased non­culprit intima­media thickness of an infarct­related artery (IRA) are associated with an altered balance of arginine metabolites. PATIENTS AND METHODS: Arginine and its metabolites were measured using liquid chromatography and tandem mass spectrometry in 100 consecutive MI patients upon admission and at 6­month follow­up. TCFA and adjacent to culprit lesion proximal and distal 10­mm segments were assessed with optical coherence tomography in the acute phase. Twenty five patients without coronary lesions on angiography served as controls. RESULTS: The C/O ratio increased 5.33 times (P <0.001), while the O/A ratio decreased 2.53 times (P <0.001) at the 6­month follow­up, as compared with the acute phase of MI. The patients with (n = 75) vs without (n = 25) TCFA had lower C/O ratio by 29% (P = 0.003), while the mean intima­media diameter of adjacent non­culprit region correlated with the follow­up O/A ratio (R = 0.337; P = 0.003). In a multivariable analysis, a higher acute phase C/O ratio was associated with a lower risk of TCFA presence (odds ratio, 0.978; 95% CI, 0.962-0.994; P = 0.006), whereas a higher follow­up O/A ratio correlated with larger intima­media diameter of the adjacent segments (ß coefficient, 0.227; 95% CI for ß coefficient, 0.045-0.409; P = 0.018). CONCLUSIONS: Enhanced arginase activity over nitric oxide synthase following ischemia was associated with the presence of TCFA in the culprit lesion, while a similar metabolic shift in the chronic phase correlated with a greater thickness of the intima­media in the adjacent non­culprit IRA segments.

γ-Tocotrienol Protects against Mitochondrial Dysfunction, Energy Deficits, Morphological Damage, and Decreases in Renal Functions after Renal Ischemia.

Ischemia-induced mitochondrial dysfunction and ATP depletion in the kidney result in disruption of primary functions and acute injury of the kidney. This study tested whether γ-tocotrienol (GTT), a member of the vitamin E family, protects mitochondrial function, reduces ATP deficits, and improves renal functions and survival after ischemia/reperfusion injury. Vehicle or GTT (200 mg/kg) were administered to mice 12 h before bilateral kidney ischemia, and endpoints were assessed at different timepoints of reperfusion. GTT treatment reduced decreases in state 3 respiration and accelerated recovery of this function after ischemia. GTT prevented decreases in activities of complexes I and III of the respiratory chain, and blocked ischemia-induced decreases in F0F1-ATPase activity and ATP content in renal cortical tissue. GTT improved renal morphology at 72 h after ischemia, reduced numbers of necrotic proximal tubular and inflammatory cells, and enhanced tubular regeneration. GTT treatment ameliorated increases in plasma creatinine levels and accelerated recovery of creatinine levels after ischemia. Lastly, 89% of mice receiving GTT and 70% of those receiving vehicle survived ischemia. Conclusions: Our data show novel observations that GTT administration improves mitochondrial respiration, prevents ATP deficits, promotes tubular regeneration, ameliorates decreases in renal functions, and increases survival after acute kidney injury in mice.

Predictive significance of selected gene mutations in relapsed and refractory chronic lymphocytic leukemia patients treated with ibrutinib.

BACKGROUND: Ibrutinib, an inhibitor of the Bruton's kinase (BTK), is characterized by high efficacy in the therapy of patients with relapsed and refractory chronic lymphocytic leukemia (RR-CLL). AIMS: To analyze the potential significance of the mutational status of selected 30 genes on the disease outcome in 45 patients with RR-CLL using custom-made gene panel and sequencing on Illumina MiSeq FGx platform. RESULTS: The highest rate of mutations was observed in TP53 (n = 18; 40.0%), NOTCH1 (n = 13; 28.8%), SF3B1 (n = 11; 24.4%), ATM (n = 7; 15.6%), MED12 (n = 6, 13.3%), CHD2 (n = 5; 11.1%), XPO1 (n = 5; 11.1%), NFKBIE (n = 5; 11.1%), BIRC3 (n = 4; 8.9%), SPEN (n = 4; 8.9%), POT1 (n = 4; 8.9%), EGR2 (n = 3; 6.7%), and RPS15 (n = 3; 6.7%). With a median observation time of 45.9 months, the median progression-free survival (PFS) and overall survival (OS) were not reached. The 36-month estimated rate of PFS and OS were 64% and 68.2%, respectively. The overall response rate was noted in 23 patients (51.1%), while twenty (44.4%) patients achieved stability. Progression was noted in 2 (4.5%) cases. Analyzed molecular factors had no impact on PFS and OS. CONCLUSION: Despite accumulation of several poor prognostic factors in our real-life cohort of heavily pretreated patients with CLL, ibrutinib treatment showed long-term clinical benefit.

Deletion of VDAC1 Hinders Recovery of Mitochondrial and Renal Functions After Acute Kidney Injury.

Voltage-dependent anion channels (VDACs) constitute major transporters mediating bidirectional movement of solutes between cytoplasm and mitochondria. We aimed to determine if VDAC1 plays a role in recovery of mitochondrial and kidney functions after ischemia-induced acute kidney injury (AKI). Kidney function decreased after ischemia and recovered in wild-type (WT), but not in VDAC1-deficient mice. Mitochondrial maximum respiration, activities of respiratory complexes and FoF1-ATPase, and ATP content in renal cortex decreased after ischemia and recovered in WT mice. VDAC1 deletion reduced respiration and ATP content in non-injured kidneys. Further, VDAC1 deletion blocked return of activities of respiratory complexes and FoF1-ATPase, and recovery of respiration and ATP content after ischemia. Deletion of VDAC1 exacerbated ischemia-induced mitochondrial fission, but did not aggravate morphological damage to proximal tubules after ischemia. However, VDAC1 deficiency impaired recovery of kidney morphology and increased renal interstitial collagen accumulation. Thus, our data show a novel role for VDAC1 in regulating renal mitochondrial dynamics and recovery of mitochondrial function and ATP levels after AKI. We conclude that the presence of VDAC1 (1) stimulates capacity of renal mitochondria for respiration and ATP production, (2) reduces mitochondrial fission, (3) promotes recovery of mitochondrial function and dynamics, renal morphology, and kidney functions, and (4) increases survival after AKI.

Protein kinase Cα mediates recovery of renal and mitochondrial functions following acute injury.

Previously, we have shown that active protein kinase Cα (PKCα) promotes recovery of mitochondrial function after injury in vitro [Nowak G & Bakajsova D (2012) Am J Physiol Renal Physiol 303, F515-F526]. This study examined whether PKCα regulates recovery of mitochondrial and kidney functions after ischemia-induced acute injury (AKI) in vivo. Markers of kidney injury were increased after bilateral ischemia and returned to normal levels in wild-type (WT) mice. Maximum mitochondrial respiration and activities of respiratory complexes and Fo F1 -ATPase decreased after ischemia and recovered in WT mice. Reperfusion after ischemia was accompanied by translocation of active PKCα to mitochondria. PKCα deletion reduced mitochondrial respiration and activities of respiratory complex I and Fo F1 -ATPase in noninjured kidneys, indicating that PKCα is essential in developing fully functional renal mitochondria. These changes in PKCα-deficient mice were accompanied by lower levels of complex I subunits (NDUFA9 and NDUFS3) and the γ-subunit of Fo F1 -ATPase. Also, lack of PKCα exacerbated ischemia-induced decreases in respiration, complex I and Fo F1 -ATPase activities, and blocked their recovery after injury, indicating a crucial role of PKCα in promoting mitochondrial recovery after AKI. Further, PKCα deletion exacerbated acetylation and succinylation of key mitochondrial proteins of energy metabolism after ischemia due to decreases in deacetylase and desuccinylase (sirtuin3 and sirtuin5) levels in renal mitochondria. Thus, our data show a novel role for PKCα in regulating levels of mitochondrial sirtuins and acetylation and succinylation of key mitochondrial proteins. We conclude that PKCα deletion: (a) affects renal physiology by decreasing mitochondrial capacity for maximum respiration; (b) blocks recovery of mitochondrial functions, renal morphology, and functions after AKI; and (c) decreases survival after AKI. ENZYMES: Protein kinase C: EC 2.7.11.13; NADH : ubiquinone reductase (H+ -translocating; complex I): EC 7.1.1.2; FoF1-ATPase (H+ -transporting two-sector ATPase): EC 7.1.2.2; Succinate : ubiquinone oxidoreductase (complex II): EC 1.3.5.1; Ubiquinol : cytochrome-c reductase (complex III): EC 7.1.1.8; Cytochrome c oxidase (complex IV): EC 1.9.3.1; NAD-dependent protein deacetylase sirtuin-3, mitochondrial: EC 2.3.1.286; NAD-dependent protein deacetylase sirtuin-5, mitochondrial: EC 3.5.1.-; Proteinase K (peptidase K): EC 3.4.21.64.

Protein kinase Cε targets respiratory chain and mitochondrial membrane potential but not F0 F1 -ATPase in renal cells injured by oxidant.

We have previously shown that protein kinase Cε (PKCε) is involved in mitochondrial dysfunction in renal proximal tubular cells (RPTC). This study examined mitochondrial targets of active PKCε in RPTC injured by the model oxidant tert-butyl hydroperoxide (TBHP). TBHP exposure augmented the levels of phosphorylated (active) PKCε in mitochondria, which suggested translocation of PKCε to mitochondria after oxidant exposure. Oxidant injury decreased state 3 respiration, adenosine triphosphate (ATP) production, ATP content, and complex I activity. Further, TBHP exposure increased ΔΨm and production of reactive oxygen species (ROS), and induced mitochondrial fragmentation and RPTC death. PKCε activation by overexpressing constitutively active PKCε exacerbated decreases in state 3 respiration, complex I activity, ATP content, and augmented RPTC death. In contrast, inhibition of PKCε by overexpressing dnPKCε mutant restored state 3 respiration, respiratory control ratio, complex I activity, ΔΨm , and ATP production and content, but did not prevent decreases in F0 F1 -ATPase activity. Inhibition of PKCε prevented oxidant-induced production of ROS and mitochondrial fragmentation, and reduced RPTC death. We conclude that activation of PKCε mediates: (a) oxidant-induced changes in ΔΨm , decreases in mitochondrial respiration, complex I activity, and ATP content; (b) mitochondrial fragmentation; and (c) RPTC death. In contrast, oxidant-induced inhibition of F0 F1 -ATPase activity is not mediated by PKCε. These results show that, in contrast to the protective effects of PKCε in the heart, PKCε activation is detrimental to mitochondrial function and viability in RPTC and mediates oxidant-induced injury.

Microenvironment-induced PIM kinases promote CXCR4-triggered mTOR pathway required for chronic lymphocytic leukaemia cell migration.

Lymph node microenvironment provides chronic lymphocytic leukaemia (CLL) cells with signals promoting their survival and granting resistance to chemotherapeutics. CLL cells overexpress PIM kinases, which regulate apoptosis, cell cycle and migration. We demonstrate that BCR crosslinking, CD40 stimulation, and coculture with stromal cells increases PIMs expression in CLL cells, indicating microenvironment-dependent PIMs regulation. PIM1 and PIM2 expression at diagnosis was higher in patients with advanced disease (Binet C vs. Binet A/B) and in those, who progressed after first-line treatment. In primary CLL cells, inhibition of PIM kinases with a pan-PIM inhibitor, SEL24-B489, decreased PIM-specific substrate phosphorylation and induced dose-dependent apoptosis in leukaemic, but not in normal B cells. Cytotoxicity of SEL24-B489 was similar in TP53-mutant and TP53 wild-type cells. Finally, inhibition of PIM kinases decreased CXCR4-mediated cell chemotaxis in two related mechanisms-by decreasing CXCR4 phosphorylation and surface expression, and by limiting CXCR4-triggered mTOR pathway activity. Importantly, PIM and mTOR inhibitors similarly impaired migration, indicating that CXCL12-triggered mTOR is required for CLL cell chemotaxis. Given the microenvironment-modulated PIM expression, their pro-survival function and a role of PIMs in CXCR4-induced migration, inhibition of these kinases might override microenvironmental protection and be an attractive therapeutic strategy in this disease.

Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury.

Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia.

Comparison of high-resolution melting analysis with direct sequencing for the detection of recurrent mutations in DNA methyltransferase 3A and isocitrate dehydrogenase 1 and 2 genes in acute myeloid leukemia patients.

Acute myeloid leukemia (AML) cells harbor frequent mutations in genes responsible for epigenetic modifications. Increasing evidence of clinical role of DNMT3A and IDH1/2 mutations highlights the need for a robust and inexpensive test to identify these mutations in routine diagnostic work-up. Herein, we compared routinely used direct sequencing method with high-resolution melting (HRM) assay for screening DNMT3A and IDH1/2 mutations in patients with AML. We show very high concordance between HRM and Sanger sequencing (100% samples for IDH2-R140 and DNMT3-R882 mutations, 99% samples for IDH1-R132 and IDH2-R172 mutations). HRM method reported no false-negative results, suggesting that it can be used for mutations screening. Moreover, HRM displayed much higher sensitivity in comparison with DNA sequencing in all assessed loci. With Sanger sequencing, robust calls were observed when the sample contained 50% of mutant DNA in the background of wild-type DNA. In marked contrast, the detection limit of HRM improved down to 10% of mutated DNA. Given the ubiquitous presence of wild-type DNA background in bone marrow aspirates and clonal variations regarding mutant allele burden, these results favor HRM as a sensitive, specific, labor-, and cost-effective tool for screening and detection of mutations in IDH1/2 and DNMT3A genes in patients with AML.

Protein kinase C-α interaction with F0F1-ATPase promotes F0F1-ATPase activity and reduces energy deficits in injured renal cells.

We showed previously that active PKC-α maintains F0F1-ATPase activity, whereas inactive PKC-α mutant (dnPKC-α) blocks recovery of F0F1-ATPase activity after injury in renal proximal tubules (RPTC). This study tested whether mitochondrial PKC-α interacts with and phosphorylates F0F1-ATPase. Wild-type PKC-α (wtPKC-α) and dnPKC-α were overexpressed in RPTC to increase their mitochondrial levels, and RPTC were exposed to oxidant or hypoxia. Mitochondrial levels of the γ-subunit, but not the α- and ß-subunits, were decreased by injury, an event associated with 54% inhibition of F0F1-ATPase activity. Overexpressing wtPKC-α blocked decreases in γ-subunit levels, maintained F0F1-ATPase activity, and improved ATP levels after injury. Deletion of PKC-α decreased levels of α-, ß-, and γ-subunits, decreased F0F1-ATPase activity, and hindered the recovery of ATP content after RPTC injury. Mitochondrial PKC-α co-immunoprecipitated with α-, ß-, and γ-subunits of F0F1-ATPase. The association of PKC-α with these subunits decreased in injured RPTC overexpressing dnPKC-α. Immunocapture of F0F1-ATPase and immunoblotting with phospho(Ser) PKC substrate antibody identified phosphorylation of serine in the PKC consensus site on the α- or ß- and γ-subunits. Overexpressing wtPKC-α increased phosphorylation and protein levels, whereas deletion of PKC-α decreased protein levels of α-, ß-, and γ-subunits of F0F1-ATPase in RPTC. Phosphoproteomics revealed phosphorylation of Ser(146) on the γ subunit in response to wtPKC-α overexpression. We concluded that active PKC-α 1) prevents injury-induced decreases in levels of γ subunit of F0F1-ATPase, 2) interacts with α-, ß-, and γ-subunits leading to increases in their phosphorylation, and 3) promotes the recovery of F0F1-ATPase activity and ATP content after injury in RPTC.

Radiation-induced alterations in mitochondria of the rat heart.

Radiation therapy for the treatment of thoracic cancers may be associated with radiation-induced heart disease (RIHD), especially in long-term cancer survivors. Mechanisms by which radiation causes heart disease are largely unknown. To identify potential long-term contributions of mitochondria in the development of radiation-induced heart disease, we examined the time course of effects of irradiation on cardiac mitochondria. In this study, Sprague-Dawley male rats received image-guided local X irradiation of the heart with a single dose ranging from 3-21 Gy. Two weeks after irradiation, left ventricular mitochondria were isolated to assess the dose-dependency of the mitochondrial permeability transition pore (mPTP) opening in a mitochondrial swelling assay. At time points from 6 h to 9 months after a cardiac dose of 21 Gy, the following analyses were performed: left ventricular Bax and Bcl-2 protein levels; apoptosis; mitochondrial inner membrane potential and mPTP opening; mitochondrial mass and expression of mitophagy mediators Parkin and PTEN induced putative kinase-1 (PINK-1); mitochondrial respiration and protein levels of succinate dehydrogenase A (SDHA); and the 70 kDa subunit of complex II. Local heart irradiation caused a prolonged increase in Bax/Bcl-2 ratio and induced apoptosis between 6 h and 2 weeks. The mitochondrial membrane potential was reduced until 2 weeks, and the calcium-induced mPTP opening was increased from 6 h up to 9 months. An increased mitochondrial mass together with unaltered levels of Parkin suggested that mitophagy did not occur. Lastly, we detected a significant decrease in succinate-driven state 2 respiration in isolated mitochondria from 2 weeks up to 9 months after irradiation, coinciding with reduced mitochondrial levels of succinate dehydrogenase A. Our results suggest that local heart irradiation induces long-term changes in cardiac mitochondrial membrane functions, levels of SDH and state 2 respiration. At any time after exposure to radiation, cardiac mitochondria are more prone to mPTP opening. Future studies will determine whether this makes the heart more susceptible to secondary stressors such as calcium overload or ischemia/reperfusion.

Protein kinase C-α interaction with iHSP70 in mitochondria promotes recovery of mitochondrial function after injury in renal proximal tubular cells.

This study determined the role of PKC-α and associated inducible heat shock protein 70 (iHSP70) in the repair of mitochondrial function in renal proximal tubular cells (RPTCs) after oxidant injury. Wild-type PKC-α (wtPKC-α) and an inactive PKC-α [dominant negative dn; PKC-α] mutant were overexpressed in primary cultures of RPTCs, and iHSP70 levels and RPTC regeneration were assessed after treatment with the oxidant tert-butylhydroperoxide (TBHP). TBHP exposure increased ROS production and induced RPTC death, which was prevented by ferrostatin and necrostatin-1 but not by cyclosporin A. Overexpression of wtPKC-α maintained mitochondrial levels of active PKC-α, reduced cell death, and accelerated proliferation without altering ROS production in TBHP-injured RPTCs. In contrast, dnPKC-α blocked proliferation and monolayer regeneration. Coimmunoprecipitation and proteomic analysis demonstrated an association between inactive, but not active, PKC-α and iHSP70 in mitochondria. Mitochondrial iHSP70 levels increased as levels of active PKC-α decreased after injury. Overexpression of dnPKC-α augmented, whereas overexpression of wtPKC-α abrogated, oxidant-induced increases in mitochondrial iHSP70 levels. iHSP70 overexpression (1) maintained mitochondrial levels of phosphorylated PKC-α, (2) improved the recovery of state 3 respiration and ATP content, (3) decreased RPTC death (an effect abrogated by cyclosporine A), and (4) accelerated proliferation after oxidant injury. In contrast, iHSP70 inhibition blocked the recovery of ATP content and exacerbated RPTC death. Inhibition of PKC-α in RPTC overexpressing iHSP70 blocked the protective effects of iHSP70. We conclude that active PKC-α maintains mitochondrial function and decreases cell death after oxidant injury. iHSP70 is recruited to mitochondria in response to PKC-α dephosphorylation and associates with and reactivates inactive PKC-α, which promotes the recovery of mitochondrial function, decreases RPTC death, and improves regeneration.

Assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase C isozymes.

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC. Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis. These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

Serum tumor necrosis factor-α and interleukin-10 levels as markers to predict outcome of patients with chronic lymphocytic leukemia in different risk groups defined by the IGHV mutation status.

Tumor necrosis factor (TNF)-α and interleukin (IL)-10 are cytokines involved in the balance between cell-mediated and humoral immunity. We investigated whether serum TNF-α and IL-10 levels have any impact on clinical outcome of patients with chronic lymphocytic leukemia (CLL). TNF-α and IL-10 levels were determined in the serum of 160 CLL patients at the time of diagnosis. The cytokine low-risk group consisted of patients with either TNF-α and IL-10 levels below their medians or those with only one elevated parameter. Both TNF-α and IL-10 levels greater than or equal to their medians defined the cytokine high-risk group. The high-risk patients presented a shorter 3-year treatment-free survival (TFS) than low-risk subjects (15 vs. 69.6 %; p < 0.0001). The high-risk group (p = 0.0002) along with high leukocyte count (p < 0.0001) and unmutated immunoglobulin heavy-chain variable region genes (p < 0.0001) independently predict the risk of progression in patients with Rai stage 0-II. Furthermore, the high-risk group had an independent prognostic impact on shorter TFS both in patients with mutated (24.3 vs. 78.2 %; p < 0.0001) and unmutated (8.2 vs. 49 %; p = 0.004) immunoglobulin heavy-chain variable region genes (IGHV) as compared to the low-risk group. The estimated 5-year overall survival (OS) of high-risk patients was shorter than those in the low-risk group (83.3 vs. 97.1 %; p = 0.003). Multivariate analysis demonstrated the cytokine high-risk group (p = 0.02) followed by Rai stage III-IV (p = 0.048) to be independent factors predicting shorter OS. At diagnosis, TNF-α and IL-10 may predict the outcome of patients with CLL.

Protein kinase C-α activation promotes recovery of mitochondrial function and cell survival following oxidant injury in renal cells.

We demonstrated that nonselective PKC activation promotes mitochondrial function in renal proximal tubular cells (RPTC) following toxicant injury. However, the specific PKC isozyme mediating this effect is unknown. This study investigated the role of PKC-α in the recovery of mitochondrial functions in oxidant-injured RPTC. Wild-type PKC-α (wtPKC-α) and inactive PKC-α mutants were overexpressed in RPTC to selectively increase or block PKC-α activation. Oxidant (tert-butyl hydroperoxidel; TBHP) exposure activated PKC-α in RPTC but decreased PKC-α levels in mitochondria following treatment. Uncoupled and state 3 respirations and activities of complexes I and IV in TBHP-injured cells decreased to 55, 44, 49, and 65% of controls, respectively. F(0)F(1)-ATPase activity and ATP content in injured RPTC decreased to 59 and 60% of controls, respectively. Oxidant exposure increased reactive oxygen species (ROS) production by 210% and induced mitochondrial fragmentation and 52% RPTC lysis. Overexpressing wtPKC-α did not block TBHP-induced ROS production but improved respiration and complex I activity, restored complex IV and F(0)F(1)-ATPase activities, promoted recovery of ATP content, blocked mitochondrial fragmentation, and reduced RPTC lysis to 14%. In contrast, inhibiting PKC-α 1) induced mitochondrial hyperpolarization and fragmentation; 2) blocked increases in ROS production; 3) prevented recovery of respiratory complexes and F(0)F(1)-ATPase activities, respiration, and ATP content; and 4) exacerbated TBHP-induced RPTC lysis. We conclude that 1) activation of PKC-α prevents mitochondrial hyperpolarization and fragmentation, decreases cell death, and promotes recovery of mitochondrial respiration and ATP content following oxidant injury in RPTC; and 2) respiratory complexes I and IV and F(0)F(1)-ATPase are targets of active PKC-α.

γ-Tocotrienol protects against mitochondrial dysfunction and renal cell death.

Oxidative stress is a major mechanism of a variety of renal diseases. Tocopherols and tocotrienols are well known antioxidants. This study aimed to determine whether γ-tocotrienol (GT3) protects against mitochondrial dysfunction and renal proximal tubular cell (RPTC) injury caused by oxidants. Primary cultures of RPTCs were injured by using tert-butyl hydroperoxide (TBHP) in the absence and presence of GT3 or α-tocopherol (AT). Reactive oxygen species (ROS) production increased 300% in TBHP-injured RPTCs. State 3 respiration, oligomycin-sensitive respiration, and respiratory control ratio (RCR) decreased 50, 63, and 47%, respectively. The number of RPTCs with polarized mitochondria decreased 54%. F0F1-ATPase activity and ATP content decreased 31 and 65%, respectively. Cell lysis increased from 3% in controls to 26 and 52% at 4 and 24 h, respectively, after TBHP exposure. GT3 blocked ROS production, ameliorated decreases in state 3 and oligomycin-sensitive respirations and F0F1-ATPase activity, and maintained RCR and mitochondrial membrane potential (ΔΨ(m)) in injured RPTCs. GT3 maintained ATP content, blocked RPTC lysis at 4 h, and reduced it to 13% at 24 h after injury. Treatment with equivalent concentrations of AT did not block ROS production and cell lysis and moderately improved mitochondrial respiration and coupling. This is the first report demonstrating the protective effects of GT3 against RPTC injury by: 1) decreasing production of ROS, 2) improving mitochondrial respiration, coupling, ΔΨ(m), and F0F1-ATPase function, 3) maintaining ATP levels, and 4) preventing RPTC lysis. Our data suggest that GT3 is superior to AT in protecting RPTCs against oxidant injury and may prove therapeutically valuable for preventing renal injury associated with oxidative stress.

Protein kinase C-epsilon activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules.

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307-F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F(0)F(1)-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F(0)F(1)-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC.