Melatonin reverses the oxidative stress and mitochondrial dysfunction caused by LETM1 silencing.

LETM1 is a mitochondrial inner-membrane protein, which is encoded by a gene present in a locus of 4p, which, in turn, is deleted in the Wolf-Hirschhorn Syndrome, and is assumed to be related to its pathogenesis. The cellular damage caused by the deletion is presumably related to oxidative stress. Melatonin has many beneficial roles in protecting mitochondria by scavenging reactive oxygen species, maintaining membrane potential, and improving functions. The aim of this study was to investigate the effects of melatonin administration to LETM1-silenced mouse embryonic fibroblast cells as a cellular model for LETM1 deficiency. We transfected mouse embryonic fibroblast cells with a pair of siRNA against LETM1 and monitored the oxidative stress and mitochondrial functions with or without melatonin addition. MnSOD expression and aconitase activity decreased and oxidized protein levels increased in LETM1-silenced cells. LETM1 suppression did not alter the expression of OXPHOS complexes, but the oxygen consumption rates decreased significantly; however, this change was not related to complex I but instead involved complex IV and complex II. Melatonin supplementation effectively normalized the parameters studied, including the oxygen consumption rate. Our findings identified a novel effect of LETM1 deficiency on cellular respiration via complex II as well as a potential beneficial role of melatonin treatment. On the other hand, these effects may be specific to the cell line used and need to be verified in other cell lines.

The Mitochondrial DNA Control Region Might Have Useful Diagnostic and Prognostic Biomarkers for Thyroid Tumors.

The literature suggests that mitochondrial DNA (mtDNA) defects are associated with a large number of diseases including cancers. The role of mtDNA variations in thyroid cancer is a highly controversial topic. Therefore, we investigated the role of mt-DNA control region (CR) variations in thyroid tumor progression and the influence of mtDNA haplogroups on susceptibility to thyroid tumors. For this purpose, in total, 108 hot thyroid nodules (HTNs), 95 cold thyroid nodules (CTNs), 48 papillary thyroid carcinoma (PTC) samples with their surrounding tissues and 104 healthy control subjects' blood samples were screened for all mtDNA CR variations using Sanger sequencing. We found that MtDNA haplogroup U was significantly associated with susceptibility to benign thyroid entities. In addition, eight single nucleotide polymorphisms (SNPs) (T146C, G185A, C194T, C295T, G16129A, T16304C, A16343G and T16362C) in the mtDNA CR were associated with the occurrence of benign and malign thyroid nodules in the Turkish population. As compared with samples taken from a healthy Turkish population and HTNs, the frequency of C7 repeats in D310 polycytosine sequence was found to be higher in CTNs and the PTC samples. In addition, the frequency of somatic mutations in mtMSI regions including T16189C and D514 CA dinucleotide repeats were found to be higher in PTC samples than benign thyroid nodules. Conversely, the frequency of somatic mutations in D310 was found to be higher in HTNs than CTNs and PTCs. In conclusion, mtDNA D310 instability does not play a role in the tumorigenesis of PTC but the results indicate that it might be used as a diagnostic clonal expansion biomarker for premalignant thyroid tumor cells. In addition, D514 CA instability might be considered as a prognostic biomarker for benign to malign transformation in thyroid tumors.

Effects of lecithin: cholesterol acyltransferase genotypes, enzyme levels, and activity on high-density lipoprotein levels.

BACKGROUND: Lecithin:cholesterol acyltransferase (LCAT) is one of the key enzymes controlling cholesterol homeostasis and plays a primary role in high-density lipoprotein cholesterol (HDL-C) maturation. OBJECTIVE: The aim of our study was to evaluate the effects of LCAT gene polymorphisms 511C/T (exon4), 4886C/T (rs5923), and 608C/T (rs5922) on LCAT enzyme level, activity, and HDL-C levels. METHODS: The study population was selected from consecutive subjects with low (<35 mg/dL) and high HDL-C levels (>65 mg/dL) seen in our lipid clinic. LCAT polymorphisms were analyzed with a restriction fragment length polymorphism assay. LCAT activity and levels were measured by colorimetric enzymatic and enzyme-linked immunoassay methods, respectively. RESULTS: The 4886C/T polymorphism was the most commonly observed variant of LCAT gene. T-allele frequencies in subjects with low (n = 50) and high (n = 50) HDL-C were 0.54 and 0.37, respectively (P = .019). TT genotype was more common among low HDL-C group (30% vs 14%, P = .05). The effects of LCAT enzyme appeared to depend on the HDL-C level. In subjects with low HDL-C, LCAT enzyme levels correlated positively with body mass index (P < .001, r = 0.544), HDL-C (P = .006, r = 0.404), triglycerides (P = .001, r = 0.487), total cholesterol (P < .001, r = 0.541), and low-density lipoprotein-cholesterol (P = .001, r = 0.477) levels. LCAT activity correlated positively with fasting glucose levels (P = .008, r = 0.390). CONCLUSION: LCAT genotype, enzyme level, and activity modulate HDL-C metabolism, particularly among subjects with low HDL-C levels.

Investigation of relationship of the mitochondrial DNA 16189 T>C polymorphism with metabolic syndrome and its associated clinical parameters in Turkish patients.

OBJECTIVE: Mitochondrial DNA (mtDNA) polymorphisms have been implicated in the pathophysiology of human diseases. Among them, a T>C nucleotide transition on the 16189 nucleotide position of mtDNA has been studied in several metabolic diseases including diabetes and obesity. In this study we aimed to investigate the association of this polymorphism among Turkish metabolic syndrome patients. DESIGN: A total of 220 cases (70 MetS patients and 150 healthy control subjects) were evaluated for their mtDNA 16189 variant by PCR-RFLP technique. In addition, clinical and biochemical variables, such as cholesterol levels, body fat percentage, insulin resistance and presence of type II diabetes, were also evaluated. RESULTS: Overall frequency of polymorphic C allele was determined as 0.19 without a significant association with type II diabetes and metabolic syndrome. This may be partly due to ethnical differences of populations studied and may also be related to other genetic and environmental factors. Moreover, there were no significant associations with biochemical variables among metabolic syndrome patients, except LDL and suppressed cortisol (sup-cortisol) levels. Low levels of LDL and sup-cortisol were significantly associated with the mtDNA 16189 variant, though the biochemical mechanism underlying this effect is not clear. CONCLUSIONS: This is the first study involving a Turkish population on the mtDNA 16189 T>C polymorphism. Further studies with larger cohorts will be needed to elucidate its relation with metabolic syndrome as well as lipid metabolism.

Prophylactic feeding with immune-enhanced diet ameliorates chemoradiation-induced gastrointestinal injury in rats.

PURPOSE: To investigate the protective effect of immune-enhanced diet (IED) on chemoradiation-induced injury of the gastrointestinal mucosa. MATERIALS AND METHODS: Forty-eight Sprague-Dawley rats were divided into control (C, n=6), irradiation (IR, n=14), fluoropyrimidine (5-FU, n=14)-treated, IR + 5-FU (n=14)-treated groups. Half of each irradiated and/or 5-FU-treated groups were previously fed with IED containing arginine, omega-3-fatty acids and RNA fragments, while the other half were fed a standard rat diet (SD) for eight days before the induction of IR or injection of 5-FU. In IR groups, whole abdominal irradiation (11 Gy) was performed with 6 MV photons. In the 5-FU groups, fluoropyrimidine (100 mg/kg) was administered intraperitoneally 30 min prior to irradiation. All animals were sacrificed on the 4th day of IR or 5-FU injection. RESULTS: Bacterial colony counts in the ceca and mesenteric lymph nodes of IED-fed rats, which have received either 5-FU and/or irradiation were significantly lower than the corresponding SD-fed groups. Morphometric results revealed that gastric, ileal and colonic injuries were less in IED-treated IR or IR + 5-FU + IED groups, as compared to SD-fed groups. However, IED did not alter DNA fragmentation ratios. CONCLUSION: Prophylactic feeding of IED has a protective effect on chemoradiation-induced gastrointestinal injury, which appears to involve the eradication of bacterial overgrowth.

Mitochondrial DNA common deletion is not associated with thyroid, breast and colorectal tumors in Turkish patients.

Recently, efforts have been focused on mitochondrial DNA changes and their relation to human cancers. Among them, a 4977 bp deletion of mitochondrial DNA, named "common deletion", has been investigated in several types of tumors, with inconsistent results. In this study, we investigated the presence of the common deletion in tissues from 25 breast, 25 colorectal and 50 thyroid tumors and in the adjacent healthy tissues from Turkish patients. Samples from healthy volunteers were also evaluated for comparison. Two PCR-based methods were used for the detection of the common deletion. First, two pairs of primers were used to amplify wild-type and deleted mtDNA. Then, a highly sensitive nested-PCR was performed, to determine low amounts of deleted genomes. By the first method, wild-type mtDNAs were observed in all samples, but a deletion was observed in only six thyroid samples, by using the nested-PCR method. In conclusion, the mitochondrial common deletion was very rare in our study group and did not appear to be not related with cancer.

Mitochondrial DNA common deletion is not associated with thyroid, breast and colorectal tumors in Turkish patients

Recently, efforts have been focused on mitochondrial DNA changes and their relation to human cancers. Among them, a 4977 bp deletion of mitochondrial DNA, named "common deletion", has been investigated in several types of tumors, with inconsistent results. In this study, we investigated the presence of the common deletion in tissues from 25 breast, 25 colorectal and 50 thyroid tumors and in the adjacent healthy tissues from Turkish patients. Samples from healthy volunteers were also evaluated for comparison. Two PCR-based methods were used for the detection of the common deletion. First, two pairs of primers were used to amplify wild-type and deleted mtDNA. Then, a highly sensitive nested-PCR was performed, to determine low amounts of deleted genomes. By the first method, wild-type mtDNAs were observed in all samples, but a deletion was observed in only six thyroid samples, by using the nested-PCR method. In conclusion, the mitochondrial common deletion was very rare in our study group and did not appear to be not related with cancer.

Erdosteine prevents colonic inflammation through its antioxidant and free radical scavenging activities.

After intracolonic administration of trinitrobenzene sulphonic acid (TNBS), Sprague-Dawley rats were treated orally either with saline or erdosteine (100 mg/kg per day), a sulfhydryl-containing antioxidant, for 3 days. On the 4th day, rats were decapitated and distal colon was removed for the macroscopic and microscopic damage scoring, for the measurement of malondialdehyde (MDA), glutathione (GSH) and collagen levels, myeloperoxidase (MPO) activity, luminol and lucigenin chemiluminescence (CL) and DNA fragmentation. Lactate dehydrogenase (LDH) activity, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and antioxidant capacity were assayed in blood samples. Colitis caused significant increases in the colonic CL values, macroscopic and microscopic damage scores, MDA and collagen levels, MPO activity and DNA fragmentation, along with a significant decrease in tissue GSH level. Similarly, serum cytokines and LDH were elevated in the saline-treated colitis group as compared with the control group. On the other hand, erdosteine treatment reversed all these biochemical indices, and histopathologic alterations induced by TNBS, suggesting that erdosteine protects the colonic tissue via its radical scavenging and antioxidant activities.

A novel approach for rapid screening of mitochondrial D310 polymorphism.

BACKGROUND: Mutations in the mitochondrial DNA (mtDNA) have been reported in a wide variety of human neoplasms. A polynucleotide tract extending from 303 to 315 nucleotide positions (D310) within the non-coding region of mtDNA has been identified as a mutational hotspot of primary tumors. This region consists of two polycytosine stretches interrupted by a thymidine nucleotide. The number of cytosines at the first and second stretches are 7 and 5 respectively, according to the GeneBank sequence. The first stretch exhibits a polymorphic length variation (6-C to 9-C) among individuals and has been investigated in many cancer types. Large-scale studies are needed to clarify the relationship between cytosine number and cancer development/progression. However, time and money consuming methods such as radioactivity-based gel electrophoresis and sequencing, are not appropriate for the determination of this polymorphism for large case-control studies. In this study, we conducted a rapid RFLP analysis using a restriction enzyme, BsaXI, for the single step simple determination of 7-C carriers at the first stretch in D310 region. METHODS: 25 colorectal cancer patients, 25 breast cancer patients and 41 healthy individuals were enrolled into the study. PCR amplification followed by restriction enzyme digestion of D310 region was performed for RFLP analysis. Digestion products were analysed by agarose gel electrophoresis. Sequencing was also applied to samples in order to confirm the RFLP data. RESULTS: Samples containing 7-C at first stretch of D310 region were successfully determined by the BsaXI RFLP method. Heteroplasmy and homoplasmy for 7-C content was also determined as evidenced by direct sequencing. Forty-one percent of the studied samples were found to be BsaXI positive. Furthermore, BsaXI status of colorectal cancer samples were significantly different from that of healthy individuals. CONCLUSION: In conclusion, BsaXI RFLP analysis is a simple and rapid approach for the single step determination of D310 polymorphism of mitochondrial DNA. This method allows the evaluation of a significant proportion of samples without the need for sequencing- and/or radioactivity-based techniques.

Co-encapsulation of two plasmids in chitosan microspheres as a non-viral gene delivery vehicle.

PURPOSE: The aims of this study are to encapsulate two different plasmid DNAs (pGL2 and pMK3) in the same microsphere structure and to investigate in vivo transfection characteristics of chitosan microspheres. Furthermore, the effect of formulation factors, such as chitosan concentration and plasmid DNA amount on in vitro properties of microspheres were studied. METHODS: Double plasmid-loaded chitosan microspheres were prepared by complex coacervation. Release studies were done in phosphate buffered saline at 37 degrees C and released plasmid DNA was determined spectrophotometrically. Integrity of plasmid DNAs was checked by agarose gel electrophoresis. For in vivo transfection studies, microspheres were injected into the muscle of the mice and expression of proteins (beta-galactosidase and luciferase) was measured. RESULTS: High encapsulation efficiency was obtained with chitosan microspheres (90%). The size of particles was about 1.15 - 1.28 m. No dependence was observed between the size and formulation variables (chitosan concentration and the amount of plasmid). After encapsulation process, integrity of two plasmids did not change. Plasmid DNAs were continuously released from chitosan microspheres. Chitosan concentrations and plasmid amounts affected in vitro release properties. After intramuscular injection of double plasmids loaded microspheres into muscle of the mice, co-expression was obtained. High beta-galactosidase and luciferase productions were determined with these microspheres after a long post-transfection period (12 weeks). CONCLUSIONS: Our results showed that two plasmids could be encapsulated in chitosan microspheres without affecting their structural and functional integrity. Thus, sustained and high protein production was obtained with these microspheres.

Preparation and in vitro transfection efficiency of chitosan microspheres containing plasmid DNA:poly(L-lysine) complexes.

PURPOSE: Studies on DNA complexes with cationic polymers are prompted by the search for nonviral DNA carriers for gene therapy. Among them, poly(L-Lysine) (PLL) has been extensively studied. On the other hand, these systems deliver DNA as a bolus without long-term release. The aims of this study were to encapsulate plasmid DNA:poly(L-lysine) (pDNA:PLL) complexes into chitosan microspheres as an alternative to the PLL based gene delivery and investigate its in vitro release and transfection characteristics as well as plasmid DNA integrity and stability against serum and DNase I challenge. METHODS: pUC18 plasmid DNA that encoded beta-galactosidase was used as a model. The microspheres were prepared by complex coacervation method and the release and in vitro transfection properties were investigated. pDNA:PLL complexes were prepared at two different mass ratios. In vitro release studies were performed at 37 +/- 0.5 degrees C and drug release was monitored both spectrophotometrically and fluorometrically. Structural integrity of the pDNA:PLL complexes were determined by Southern blotting analysis. Protective effect of encapsulation of pDNA:PLL complexes against DNase I and serum treatment were also studied. In vitro transfection studies were performed by using 3T3 cell line. RESULTS: According to our in vitro release data, the mass ratio of pDNA:PLL significantly affected the release of pDNA:PLL complexes from chitosan microspheres, and the structure of the plasmid DNA did not change during the experiments. pDNA:PLL-loaded chitosan microspheres indicated high stability against fetal bovine serum and DNase I treatment for a week. In vitro transfection data showed that pDNA:PLL-loaded chitosan microspheres could be effectively transfected 3T3 cells in vitro. CONCLUSION: As a conclusion, pDNA:PLL complexes could be encapsulated into chitosan microspheres with maintaining their structural and functional integrity and this system may be a good alternative for polycation based gene carriers.

Controlled release of interleukin-2 from chitosan microspheres.

Chitosan microspheres were evaluated for sustained-release of recombinant human interleukin-2 (rIL-2) in this study. In addition, the effects of different formulation factors, such as chitosan and protein concentrations, the volume of sodium sulfate solution, addition technique of rIL-2, and presence of glutaraldehyde during the encapsulation process, on microsphere characteristics were investigated. Chitosan microspheres containing rIL-2 were prepared by using the precipitation technique. The average diameter of microspheres was between 1.11-1.59 microm. Recombinant IL-2 encapsulation efficiency in these microspheres was high (75-98%). Formulation factors had no effect on the microsphere size. Recombinant IL-2 had been released from chitosan microspheres over a period of 3 months. The encapsulated rIL-2 remained biologically active and could be completely recovered from the release medium. Briefly, rIL-2 was released from chitosan microspheres in a sustained manner. The efficacy of rIL-2 loaded chitosan microspheres was studied using two model cells, HeLa and L-strain cell lines. Chitosan microspheres were added to the cells at different concentrations, and the amount of rIL-2 was assayed using the ELISA kit. Cell culture studies indicated that microspheres were uptaken by cells, and rIL-2 was released from the microspheres. Cellular uptake of rIL-2-loaded microspheres was dose dependent. It can be said that chitosan microsphere is a suitable carrier for rIL-2 delivery.