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OBJECTIVE: We sought to investigate the biological effects of pre-reperfusion treatments of the liver after warm and cold ischemic injuries in a porcine donation after circulatory death model. SUMMARY OF BACKGROUND DATA: Donation after circulatory death represents a severe form of liver ischemia and reperfusion injury that has a profound impact on graft function after liver transplantation. METHODS: Twenty donor pig livers underwent 60 minutes of in situ warm ischemia after circulatory arrest and 120 minutes of cold static preservation prior to simulated transplantation using an ex vivo perfusion machine. Four reperfusion treatments were compared: Control-Normothermic (N), Control- Subnormothermic (S), regulated hepatic reperfusion (RHR)-N, and RHR-S (n = 5 each). The biochemical, metabolic, and transcriptomic profiles, as well as mitochondrial function were analyzed. RESULTS: Compared to the other groups, RHR-S treated group showed significantly lower post-reperfusion aspartate aminotransferase levels in the reperfusion effluent and histologic findings of hepatocyte viability and lesser degree of congestion and necrosis. RHR-S resulted in a significantly higher mitochondrial respiratory control index and calcium retention capacity. Transcriptomic profile analysis showed that treatment with RHR-S activated cell survival and viability, cellular homeostasis as well as other biological functions involved in tissue repair such as cytoskeleton or cytoplasm organization, cell migration, transcription, and microtubule dynamics. Furthermore, RHR-S inhibited organismal death, morbidity and mortality, necrosis, and apoptosis. CONCLUSION: Subnormothermic RHR mitigates IRI and preserves hepatic mitochondrial function after warm and cold hepatic ischemia. This organ resuscitative therapy may also trigger the activation of protective genes against IRI. Sub- normothermic RHR has potential applicability to clinical liver transplantation.
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Preservação de Órgãos , Transcriptoma , Suínos , Animais , Preservação de Órgãos/métodos , Fígado/patologia , Reperfusão , Isquemia , Necrose/metabolismo , Necrose/patologiaRESUMO
Control of myocardial energetics by Ca2+ signal propagation to the mitochondrial matrix includes local Ca2+ delivery from sarcoplasmic reticulum (SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane (IMM) Ca2+ uniporter (mtCU). mtCU activity in cardiac mitochondria is relatively low, whereas the IMM surface is large, due to extensive cristae folding. Hence, stochastically distributed mtCU may not suffice to support local Ca2+ transfer. We hypothesized that mtCU concentrated at mitochondria-SR associations would promote the effective Ca2+ transfer. mtCU distribution was determined by tracking MCU and EMRE, the proteins essential for channel formation. Both proteins were enriched in the IMM-outer mitochondrial membrane (OMM) contact point submitochondrial fraction and, as super-resolution microscopy revealed, located more to the mitochondrial periphery (inner boundary membrane) than inside the cristae, indicating high accessibility to cytosol-derived Ca2+ inputs. Furthermore, MCU immunofluorescence distribution was biased toward the mitochondria-SR interface (RyR2), and this bias was promoted by Ca2+ signaling activity in intact cardiomyocytes. The SR fraction of heart homogenate contains mitochondria with extensive SR associations, and these mitochondria are highly enriched in EMRE. Size exclusion chromatography suggested for EMRE- and MCU-containing complexes a wide size range and also revealed MCU-containing complexes devoid of EMRE (thus disabled) in the mitochondrial but not the SR fraction. Functional measurements suggested more effective mtCU-mediated Ca2+ uptake activity by the mitochondria of the SR than of the mitochondrial fraction. Thus, mtCU "hot spots" can be formed at the cardiac muscle mitochondria-SR associations via localization and assembly bias, serving local Ca2+ signaling and the excitation-energetics coupling.
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Canais de Cálcio/metabolismo , Miocárdio/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Sinalização do Cálcio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Membranas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismoRESUMO
Mitochondrial Ca2+ uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca2+ uptake and our current understanding of mitochondrial Ca2+ homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca2+ uniporter complex.
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Canais de Cálcio/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/efeitos dos fármacos , Metabolismo Energético , Homeostase , Humanos , Mitocôndrias/metabolismoRESUMO
Mitochondrial Ca(2+) homeostasis, the Ca(2+) influx-efflux balance, is responsible for the control of numerous cellular functions, including energy metabolism, generation of reactive oxygen species, spatiotemporal dynamics of Ca(2+) signaling, and cell growth and death. Recent discovery of the molecular identity of the mitochondrial Ca(2+) uniporter (MCU) provides new possibilities for application of genetic approaches to study the mitochondrial Ca(2+) influx mechanism in various cell types and tissues. In addition, the subsequent discovery of various auxiliary subunits associated with MCU suggests that mitochondrial Ca(2+) uptake is not solely regulated by a single protein (MCU), but likely by a macromolecular protein complex, referred to as the MCU-protein complex (mtCUC). Moreover, recent reports have shown the potential role of MCU posttranslational modifications in the regulation of mitochondrial Ca(2+) uptake through mtCUC. These observations indicate that mtCUCs form a local signaling complex at the inner mitochondrial membrane that could significantly regulate mitochondrial Ca(2+) handling, as well as numerous mitochondrial and cellular functions. In this review we discuss the current literature on mitochondrial Ca(2+) uptake mechanisms, with a particular focus on the structure and function of mtCUC, as well as its regulation by signal transduction pathways, highlighting current controversies and discrepancies.
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Canais de Cálcio/metabolismo , Sinalização do Cálcio , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Canais de Cálcio/química , Canais de Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Homeostase , Humanos , Ativação do Canal Iônico , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Subunidades Proteicas , Processamento Pós-Transcricional do RNA , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of Gq-protein coupled receptor (GqPCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is one of the GqPCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, ßI, ßII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (<30 s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCßI and PKCßII, to the PM (<30 s), with sustained membrane localization. One novel PKC (nPKC), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30 s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, ß and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.
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Membrana Celular/metabolismo , Proteína Quinase C/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Transdução de Sinais/fisiologia , Células HEK293 , Humanos , Isoformas de Proteínas/metabolismo , Transporte Proteico/fisiologiaRESUMO
BACKGROUND & OBJECTIVES: Current therapy for leishmaniasis is limited and unsatisfactory. Amphotericin B, a second-line treatment is gradually replacing antimonials, the first-line treatment and is used as the preferred treatments in some regions. Though, presently it is the only drug with highest cure rate, its use is severely restricted by its acute toxicity. In the present study novel lipid-amphotericin B formulations with lower toxicity than the parent drug were evaluated for the treatment of visceral leishmaniasis (VL) in a mouse model. METHODS: The toxicity and therapeutic efficacy of a new amphiphilic formulation of amphotericin B (Kalsome10) was compared to that of amphotericin B deoxycholate (Fungizone) in a mouse model of VL using quantitative real-time PCR (qRT-PCR). RESULTS: The toxicity of amphotericin B was significantly less with liposomal formulation as compared to the deoxycholate form, evidenced by reduced nephrotoxicity and higher tolerated dose in BALB/c mice. The therapeutic efficacy was evaluated by quantitative real time (RT) PCR using primers highly specific for the ITS region of Leishmania donovani. There was reduction in parasite load by 2 log unit after 7 days of treatment and finally resulting in complete clearance of parasite from infected mice after 30 days of treatment with Kalsome10. INTERPRETATION & CONCLUSIONS: This new formulation showed a favourable safety profile and better efficacy when compared to conventional amphotericin B. If production cost is kept low, it may prove to be a feasible alternative to conventional amphotericin B.
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Anfotericina B/administração & dosagem , Leishmaniose Visceral/tratamento farmacológico , Lipossomos/administração & dosagem , Anfotericina B/efeitos adversos , Anfotericina B/química , Animais , Modelos Animais de Doenças , Humanos , Leishmaniose Visceral/patologia , Lipossomos/efeitos adversos , Lipossomos/química , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Miltefosine (MIL), an alkylphospholipid, is the first orally administrable anti-leishmanial drug. But due to its long half-life, miltefosine is highly vulnerable for resistance. Hence it is important to understand the mechanism of resistance and to elucidate its action on Leishmania. Here we investigate the miltefosine induced process of programmed cell death in wild type (miltefosine sensitive) and in laboratory generated resistant strains of Leishmania donovani. Results indicate that miltefosine induced apoptosis like death in a time and dose dependent manner in wild-type cells, but not in MIL-resistant cell line. The miltefosine resistant cells remained protected against miltefosine-induced loss of mitochondrial membrane potential, gradual ATP loss and cytochrome C release from mitochondria into the cytosol. Comparative transcriptomic study showed significantly increased expression of FeSODA and SIR2 genes, putatively involved in oxidative stress associated apoptotic cell death. We hypothesize that oxidative stress mediated apoptosis as an alternative mechanism of miltefosine resistance.
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Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Antiprotozoários/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Perfilação da Expressão Gênica , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmaniose Visceral/tratamento farmacológico , Macrófagos/parasitologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Fosforilcolina/farmacologia , Fosforilcolina/uso terapêutico , Sirtuínas/genética , Sirtuínas/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Visceral leishmaniasis (VL) remains a major health problem in old world, and India accounts for half of the world burden. The widespread emergence of resistance to standard drug in India poses a major obstacle in the control of leishmaniasis. Post-Kala-Azar dermal leishmaniasis (PKDL) is considered as main source of drug resistance. Experimental data indicate that resistance against newer drugs is also imminent. Therefore, in vitro studies were carried out to test minimum parasiticidal concentration of five conventional and newly introduced anti-leishmanial drugs against 20 field isolates of Leishmania donovani obtained from visceral and post-Kala-Azar dermal leishmaniasis patients of India. Study revealed wide range of variation in minimum inhibitory concentration of sodium antimony gluconate (SAG). PKDL isolates displayed significantly lower susceptibility to SAG and miltefosine than VL isolates with P value of 0.0006 and 0.0243, respectively. All clinical isolates had higher IC(50) value for paromomycin and miltefosine as compared to reference strain indicating their vulnerability to develop unresponsiveness. However, isolates were uniformly susceptible to pentamidine and amphotericin B. The results of gene expression analysis of AQP1 were largely in agreement with phenotypic drug sensitivity results. Interestingly, significant down-regulation of AQP1 was observed in PKDL isolates as compared to VL isolates indicating their increased propensity for drug unresponsiveness. However, no significant difference in mRNA expression of LdMT and LdRos3 gene was found for two groups. The present study unravels valuable baseline scientific data showing variation in the drug susceptibility pattern in the L. donovani isolates. The information might have impact on the management and control of Indian visceral leishmaniasis.
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Antiprotozoários/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Animais , Aquaporina 1/biossíntese , Perfilação da Expressão Gênica , Humanos , Índia , Concentração Inibidora 50 , Leishmania donovani/isolamento & purificação , Testes de Sensibilidade Microbiana , Testes de Sensibilidade ParasitáriaRESUMO
MCU is widely recognized as a responsible gene for encoding a pore-forming subunit of highly mitochondrial-specific and Ca 2+ -selective channel, mitochondrial Ca 2+ uniporter complex (mtCUC). Here, we report a novel short variant derived from the MCU gene (termed MCU-S) which lacks mitochondria-targeted sequence and forms a Ca 2+ - permeable channel outside of mitochondria. MCU-S was ubiquitously expressed in all cell-types/tissues, with particularly high expression in human platelets. MCU-S formed Ca 2+ channels at the plasma membrane, which exhibited similar channel properties to those observed in mtCUC. MCU-S channels at the plasma membrane served as an additional Ca 2+ influx pathway for platelet activation. Our finding is completely distinct from the originally reported MCU gene function and provides novel insights into the molecular basis of MCU variant-dependent cellular Ca 2+ handling.
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Mitochondrial calcium (Ca2+) plays a key role in regulating normal cardiac function. A physiological increase in mitochondrial matrix calcium [Ca2+]m drives mitochondrial ATP production to meet the high-energy demands during excitation-contraction coupling. However, a pathological increase in [Ca2+]m leads to increased oxidative stress, impaired bioenergetics, and the opening of mitochondrial permeability transition pore (mPTP), a hallmark of the failing heart. Therefore, a better understanding of the [Ca2+]m handling and its role in heart function and dysfunction is of great importance. Here, we describe a detailed protocol for measuring mitochondrial Ca2+ handling in the isolated functionally intact mitochondria from cardiac tissue of the guinea pig.
Assuntos
Cálcio , Proteínas de Transporte da Membrana Mitocondrial , Animais , Cobaias , Coração , Mitocôndrias Cardíacas , Poro de Transição de Permeabilidade MitocondrialRESUMO
Background: Hypothermia (H), cardioplegia (CP), and both combined (HCP) are known to be protective against myocardial ischemia reperfusion (IR) injury. Mitochondria have molecular signaling mechanisms that are associated with both cell survival and cell death. In this study, we investigated the dynamic changes in proapoptotic and prosurvival signaling pathways mediating H, CP, or HCP-induced protection of mitochondrial function after acute myocardial IR injury. Methods: Rats were divided into five groups. Each group consists of 3 subgroups based on a specific reperfusion time (5, 20, or 60 min) after a 25-min global ischemia. The time control (TC) groups were not subjected to IR but were perfused with 37 °C Krebs-Ringer's (KR) buffer, containing 4.5 mM K+, in a specific perfusion protocol that corresponded with the duration of each IR protocol. The IR group (control) was perfused for 20 min with KR, followed by 25-min global ischemia, and then KR reperfusion for 5, 20, or 60 min. The treatment groups were exposed to 17 °C H, 37 °C CP (16 mM K+), or HCP (17 °C + CP) for 5 min before ischemia and for 2 min on reperfusion before switching to 37 °C KR perfusion for the remainder of each of the reperfusion times. Cardiac function and mitochondrial redox state (NADH/FAD) were monitored online in the ex vivo hearts before, during, and after ischemia. Mitochondria were isolated at the end of each specified reperfusion time, and changes in O2 consumption, membrane potential (ΔΨ m), and Ca2+ retention capacity (CRC) were assessed using complex I and complex II substrates. In another set of hearts, mitochondrial and cytosolic fractions were isolated after a specified reperfusion time to conduct western blot assays to determine hexokinase II (HKII) and Bax binding/translocation to mitochondria, cytosolic pAkt levels, and cytochrome c (Cyto-c) release into the cytosol. Results: H and HCP were more protective of mitochondrial integrity and, concomitantly, cardiac function than CP alone; H and HCP improved post-ischemic cardiac function by (1) maintaining mitochondrial bioenergetics, (2) maintaining HKII binding to mitochondria with an increase in pAkt levels, (3) increasing CRC, and (4) decreasing Cyto-c release during reperfusion. Bax translocation/binding to mitochondria was unaffected by any treatment, regardless of cardiac functional recovery. Conclusions: Hypothermia preserved mitochondrial function and cardiac function, in part, by maintaining mitochondrial bioenergetics, by retaining HKII binding to mitochondria via upstream pAkt, and by reducing Cyto-c release independently of Bax binding to mitochondria.
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Hipotermia , Traumatismo por Reperfusão Miocárdica , Animais , Metabolismo Energético , Hexoquinase/metabolismo , Hipotermia/metabolismo , Isquemia/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos , Reperfusão , Proteína X Associada a bcl-2/metabolismoRESUMO
Nearly 2 decades since its discovery as one of the genes responsible for the Wolf-Hirschhorn Syndrome (WHS), the primary function of the leucine-zipper EF-hand containing transmembrane 1 (LETM1) protein in the inner mitochondrial membrane (IMM) or the mechanism by which it regulates mitochondrial Ca2+ handling is unresolved. Meanwhile, LETM1 has been associated with the regulation of fundamental cellular processes, such as development, cellular respiration and metabolism, and apoptosis. This mini-review summarizes the diversity of cellular functions impacted by LETM1 and highlights the multiple roles of LETM1 in health and disease.
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BACKGROUND & OBJECTIVES: India carries approximately 50 per cent of the global burden of visceral leishmaniasis and majority of patients from the poor, rural communities of Bihar State. Zinc is an essential trace element and its relevance for proper functioning of the entire immune system is already well documented. Though low serum zinc levels have been reported in many parasitic diseases, limited information is available regarding zinc status in human leishmaniasis. We investigated to define the relationship between zinc level in visceral leishmaniasis (VL) patients in endemic and non-endemic regions. METHODS: Venous blood was collected from 88 patients, 16 parasitologically confirmed VL, 35 healthy controls from endemic area (Bihar) and 37 healthy urban controls from non-endemic area, Delhi. In all the three groups, levels of serum albumin, total protein (markers of nutritional status) and zinc were estimated by colorimetric methods. RESULTS: Serum zinc levels were found to be significantly lower (P<0.001) in VL patients than non-endemic controls. The serum zinc levels in VL endemic controls were also significantly lower (P<0.001) than non- endemic controls, but these values were not statistically significantly different from VL patients. However, all samples from Bihar (VL patients and controls) had lower serum zinc levels than non-endemic controls from Delhi. INTERPRETATION & CONCLUSION: Low serum Zn levels, in healthy subjects from Bihar and more significantly in VL patients of this region, are possibly associated with vulnerability and endemicity of visceral leishmaniasis in the region. Further studies need to be done to assess the role of oral zinc supplementation in better management and prevention of VL, particularly in endemic areas.
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Leishmaniose Visceral/sangue , Zinco/sangue , Adulto , Animais , Criança , Feminino , Humanos , Índia/epidemiologia , Leishmania , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/prevenção & controle , Masculino , Pobreza , População Rural , População Urbana , Adulto JovemRESUMO
Mitochondrial Ca2+ handling is accomplished by balancing Ca2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca2+ buffering in the matrix and Ca2+ efflux mainly via Ca2+ ion exchangers, such as the Na+/Ca2+ exchanger (NCLX) and the Ca2+/H+ exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca2+ load via repetitive application of a finite CaCl2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca2+ efflux at different matrix Ca2+ loads was revealed by inhibiting Ca2+ uptake or reuptake with Ru360 after increasing number of CaCl2 boluses. In Na+-free experimental buffer and with Ca2+ uptake inhibited, the rate of Ca2+ efflux and steady-state free matrix Ca2+ [mCa2+]ss increased as the number of administered CaCl2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca2+ buffering while maintaining a constant [mCa2+]ss, decreased the rate of Ca2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca2+ buffering rate and decreased [mCa2+]ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8â¼6.9 caused a twofold increase in the Ca2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4â¼7.5 did not change the Ca2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca2+ efflux. The results indicate that Ca2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca2+. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca2+ buffering system and the matrix free Ca2+ concentration.
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Radiation therapy is received by over half of all cancer patients. However, radiation doses may be constricted due to normal tissue side effects. In thoracic cancers, including breast and lung cancers, cardiac radiation is a major concern in treatment planning. There are currently no biomarkers of radiation-induced cardiotoxicity. Complex genetic modifiers can contribute to the risk of radiation-induced cardiotoxicities, yet these modifiers are largely unknown and poorly understood. We have previously reported the SS (Dahl salt-sensitive/Mcwi) rat strain is a highly sensitized model of radiation-induced cardiotoxicity compared to the more resistant Brown Norway (BN) rat strain. When rat chromosome 3 from the resistant BN rat strain is substituted into the SS background (SS.BN3 consomic), it significantly attenuates radiation-induced cardiotoxicity, demonstrating inherited genetic variants on rat chromosome 3 modify radiation sensitivity. Genes involved with mitochondrial function were differentially expressed in the hearts of SS and SS.BN3 rats 1 week after radiation. Here we further assessed differences in mitochondria-related genes between the sensitive SS and resistant SS.BN3 rats. We found mitochondrial-related gene expression differed in untreated hearts, while no differences in mitochondrial morphology were seen 1 week after localized heart radiation. At 12 weeks after localized cardiac radiation, differences in mitochondrial complex protein expression in the left ventricles were seen between the SS and SS.BN3 rats. These studies suggest that differences in mitochondrial gene expression caused by inherited genetic variants may contribute to differences in sensitivity to cardiac radiation.
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BACKGROUND: The contamination with Entamoeba histolytica, Giardia lamblia, and Salmonella spp. in drinking water is the most prevalent in Indian subcontinent, but often difficult to detect all these pathogens from the drinking water. MATERIALS AND METHODS: A multiplex polymerase chain reaction (mPCR) method was developed to detect contamination of municipality-supplied drinking water with E. histolytica, G. lamblia, and Salmonella spp. The primers were designed to target small subunit of 16S rRNA type gene of E. histolytica and G. lamblia, and invasive A gene of Salmonella typhimurium. The optimized mPCR assay was applied on 158 municipality-supplied drinking water samples collected from Delhi. RESULTS: Out of total 158 water samples, 89 (56.32%) were found positive for the targeted pathogens by mPCR while conventional methods could be detected only in 11 (6.96%) samples. The mPCR assay showed 100% sensitivity and specificity for these pathogens in comparison with culture and microscopic detection. Of the 89 mPCR-positive samples, G. lamblia, E. histolytica, and Salmonella spp. were present in 35 (22.15%), 26 (16.45%), and 28 (17.72%), respectively. Nine (5.69%) samples were positive for both E. histolytica and G. lamblia, 10 (6.32%) were positive for G. lamblia and Salmonella spp., and 8 (5.06%) had Salmonella spp. and E. histolytica. Nonetheless, 3 (1.89%) samples were positive for all three pathogens. CONCLUSIONS: The present assay is an alternative to conventional methods to serve as highly sensitive, specific, and economical means for water quality surveillance to detect the outbreak caused by E. histolytica, G. lamblia, and Salmonella spp. pathogens.
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Regulation of mitochondrial free Ca2+ is critically important for cellular homeostasis. An increase in mitochondrial matrix free Ca2+ concentration ([Ca2+]m) predisposes mitochondria to opening of the permeability transition pore (mPTP). Opening of the pore can be delayed by cyclosporin A (CsA), possibly by inhibiting cyclophilin D (Cyp D), a key regulator of mPTP. Here, we report on a novel mechanism by which CsA delays mPTP opening by enhanced sequestration of matrix free Ca2+. Cardiac-isolated mitochondria were challenged with repetitive CaCl2 boluses under Na+-free buffer conditions with and without CsA. CsA significantly delayed mPTP opening primarily by promoting matrix Ca2+ sequestration, leading to sustained basal [Ca2+]m levels for an extended period. The preservation of basal [Ca2+]m during the CaCl2 pulse challenge was associated with normalized NADH, matrix pH (pHm), and mitochondrial membrane potential (ΔΨm). Notably, we found that in PO43- (Pi)-free buffer condition, the CsA-mediated buffering of [Ca2+]m was abrogated, and mitochondrial bioenergetics variables were concurrently compromised. In the presence of CsA, addition of Pi just before pore opening in the Pi-depleted condition reinstated the Ca2+ buffering system and rescued mitochondria from mPTP opening. This study shows that CsA promotes Pi-dependent mitochondrial Ca2+ sequestration to delay mPTP opening and, concomitantly, maintains mitochondrial function.
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Ciclosporina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Soluções Tampão , Cálcio/metabolismo , Ciclosporina/metabolismo , Metabolismo Energético , Feminino , Cobaias , Coração/efeitos dos fármacos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Miocárdio/metabolismo , Espécies Reativas de OxigênioRESUMO
Genomic amplification of 3q26.2 locus leads to the increased expression of microRNA 551b-3p (miR551b-3p) in triple-negative breast cancer (TNBC). Our results demonstrate that miR551b-3p translocates to the nucleus with the aid of importin-8 (IPO8) and activates STAT3 transcription. As a consequence, miR551b upregulates the expression of oncostatin M receptor (OSMR) and interleukin-31 receptor-α (IL-31RA) as well as their ligands OSM and IL-31 through STAT3 transcription. We defined this set of genes induced by miR551b-3p as the "oncostatin signaling module," which provides oncogenic addictions in cancer cells. Notably, OSM is highly expressed in TNBC, and the elevated expression of OSM associates with poor outcome in estrogen-receptor-negative breast cancer patients. Conversely, targeting miR551b with anti-miR551b-3p reduced the expression of the OSM signaling module and reduced tumor growth, as well as migration and invasion of breast cancer cells.
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Progressão da Doença , MicroRNAs/metabolismo , Oncostatina M/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Animais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos Nus , MicroRNAs/genética , Terapia de Alvo Molecular , Invasividade Neoplásica , Fator de Transcrição STAT3/metabolismo , Transcrição Gênica , Ativação Transcricional/genética , Regulação para Cima/genética , beta Carioferinas/metabolismoRESUMO
Leishmaniasis is a parasitic disease caused by hemoflagellate, Leishmania spp. The parasite is transmitted by the bite of an infected female phlebotomine sandfly. The disease is prevalent throughout the world and in at least 88 countries. Nearly 25 compounds are reported to have anti-leishmanial effects but not all are in use. The pentavalent antimony compounds have remained mainstay for nearly 75 years. Pentavalent antimony is a prodrug that is reduced by glutathione to active trivalent species catalyzed by thiol-dependent-reductase. However, emergence of resistance led to the use of other compounds--amphotericin B, pentamidine, paromomycin, allopurinol etc. Amphotericin B, an antifungal macrolide polyene is characterized by the hydrophilic polyhydroxyl and hydrophobic polyene faces on it long axis. Presently, it is the only drug with highest cure rate. It acts on membrane sterols resulting in parasite cell lysis. Its lipid formulations have been developed to minimize side effects. Other anti-fungals like ketoconazole, fluconazole and terbinafine are found less effective. Recently, anticancer alkylphosphocholines have been found most effective oral compounds. These act as membrane synthetic ether-lipid analogues, and consist of alkyl chains in the lipid portions. Most promising of these are miltefosine (hexadecylphosphocholine), edelfosine (ET-18-OCH(3)) and ilmofosine (BM 41.440). However, the recent focus has been on identifying newer therapeutic targets in the parasite such as DNA topoisomerases. The present review describes the current understanding of different drugs against leishmaniasis, their chemistry, mode of action and the mechanism of resistance in the parasite. Future perspectives in the area of new anti-leishmanial drug targets are also enumerated. However, due to the vastness of the topic main emphasis is given on visceral leishmaniasis.