Silver nanoparticles: Advanced and promising technology in diabetic wound therapy.

Wounds associated with diabetes mellitus are the most severe co-morbidities, which could be progressed to cause cell necrosis leading to amputation. Statistics on the recent status of the diabetic wounds revealed that the disease affects 15% of diabetic patients, where 20% of them undergo amputation of their limb. Conventional therapies are found to be ineffective due to changes in the molecular architecture of the injured area, urging novel deliveries for effective treatment. Therefore, recent researches are on the development of new and effective wound care materials. Literature is evident in providing potential tools in topical drug delivery for wound healing under the umbrella of nanotechnology, where nano-scaffolds and nanofibers have shown promising results. The nano-sized particles are also known to promote healing of wounds by facilitating proper movement through the healing phases. To date, focuses have been made on the efficacy of silver nanoparticles (AgNPs) in treating the diabetic wound, where these nanoparticles are known to exploit potential biological properties in producing anti-inflammatory and antibacterial activities. AgNPs are also known to activate cellular mechanisms towards the healing of chronic wounds; however, associated toxicities of AgNPs are of great concern. This review is an attempt to illustrate the use of AgNPs in wound healing to facilitate this delivery system in bringing into clinical applications for a superior dressing and treatment over wounds and ulcers in diabetes patients.

Anti-cancer activity of an osthole derivative, NBM-T-BMX-OS01: targeting vascular endothelial growth factor receptor signaling and angiogenesis.

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.

Histone demethylase RBP2 promotes lung tumorigenesis and cancer metastasis.

The retinoblastoma binding protein RBP2 (KDM5A) is a histone demethylase that promotes gastric cancer cell growth and is enriched in drug-resistant lung cancer cells. In tumor-prone mice lacking the tumor suppressor gene RB or MEN1, genetic ablation of RBP2 can suppress tumor initiation, but the pathogenic breadth and mechanistic aspects of this effect relative to human tumors have not been defined. Here, we approached this question in the context of lung cancer. RBP2 was overexpressed in human lung cancer tissues where its depletion impaired cell proliferation, motility, migration, invasion, and metastasis. RBP2 oncogenicity relied on its demethylase and DNA-binding activities. RBP2 upregulated expression of cyclins D1 and E1 while suppressing the expression of cyclin-dependent kinase inhibitor p27 (CDKN1B), each contributing to RBP2-mediated cell proliferation. Expression microarray analyses revealed that RBP2 promoted expression of integrin-ß1 (ITGB1), which is implicated in lung cancer metastasis. Mechanistic investigations established that RBP2 bound directly to the p27, cyclin D1, and ITGB1 promoters and that exogenous expression of cyclin D1, cyclin E1, or ITGB1 was sufficient to rescue proliferation or migration/invasion, respectively. Taken together, our results establish an oncogenic role for RBP2 in lung tumorigenesis and progression and uncover novel RBP2 targets mediating this role.

NBM-HD-1: A Novel Histone Deacetylase Inhibitor with Anticancer Activity.

HDAC inhibitors (HDACis) have been developed as promising anticancer agents in recent years. In this study, we synthesized and characterized a novel HDACi, termed NBM-HD-1. This agent was derived from the semisynthesis of propolin G, isolated from Taiwanese green propolis (TGP), and was shown to be a potent suppressor of tumor cell growth in human breast cancer cells (MCF-7 and MDA-MB-231) and rat glioma cells (C6), with an IC(50) ranging from 8.5 to 10.3 µM. Western blot demonstrated that levels of p21((Waf1/Cip1)), gelsolin, Ac-histone 4, and Ac-tubulin markedly increased after treatment of cancer cells with NBM-HD-1. After NBM-HD-1 treatment for 1-4 h, p-PTEN and p-AKT levels were markedly decreased. Furthermore, we also found the anticancer activities of NBM-HD-1 in regulating cell cycle regulators. Treatment with NBM-HD-1, p21((Waf1/Cip1)) gene expression had markedly increased while cyclin B1 and D1 gene expressions had markedly decreased. On the other hand, we found that NBM-HD-1 increased the expressions of tumor-suppressor gene p53 in a dose-dependent manner. Finally, we showed that NBM-HD-1 exhibited potent antitumor activity in a xenograft model. In conclusion, this study demonstrated that this compound, NBM-HD-1, is a novel and potent HDACi with anticancer activity in vitro and in vivo.

Role of reactive oxygen species-elicited apoptosis in the pathophysiology of mitochondrial and neurodegenerative diseases associated with mitochondrial DNA mutations.

A wide spectrum of pathogenic mutations of mitochondrial DNA (mtDNA) has been demonstrated to cause mitochondrial dysfunction and overproduction of reactive oxygen species (ROS), in relation to mitochondrial and neurodegenerative diseases. Our previous studies have shown that large-scale deletions of mtDNA not only serve as an indicator of oxidative damage, but also result in greater susceptibility of human cells to apoptosis triggered by UV irradiation and other apoptotic stimuli. In this review, we focus on the involvement of mtDNA-mutation-associated oxidative stress and susceptibility to apoptosis in the pathophysiology of mitochondrial and neurodegenerative diseases. Different lines of research have provided concordant data to suggest that the mtDNA-mutation-elicited energy insufficiency and enhanced oxidative stress and damage lead to cell dysfunction, and increase the susceptibility of affected cells to apoptosis in patients with these diseases. Moreover, accumulating experimental evidence has shown that antioxidant therapy is a good strategy for decreasing intracellular ROS and alleviating oxidative-stress-induced apoptosis in cells of patients that harbor pathogenic mtDNA mutations.

Activation of PKCdelta and ERK1/2 in the sensitivity to UV-induced apoptosis of human cells harboring 4977 bp deletion of mitochondrial DNA.

The 4977 bp deletion of mitochondrial DNA (mtDNA), often found in patients with chronic progressive external ophthalmoplegia (CPEO), has been demonstrated to increase the susceptibility to apoptosis of human cells. We investigated the mechanism underlying the apoptotic susceptibility of the Delta4977 cybrid harboring about 80% 4977 bp-deleted mtDNA. The production of hydrogen peroxide (H(2)O(2)) and phosphorylation of PKCdelta and ERK1/2 were increased in the Delta4977 cybrid, which was more susceptible to UV-induced apoptosis. Moreover, treatment with N-acetyl-l-cysteine (NAC) or blocking of activation of PKCdelta by rottlerin or PKCdelta-siRNA, and inhibition of ERK1/2 by PD98059 or ERK1/2-siRNA significantly attenuated the susceptibility of the Delta4977 cybrid to apoptosis. Furthermore, the increase of PKCdelta expression in the Delta4977 cybrid also amplified the apoptotic signal through caspase 3-mediated proteolytic activation of PKCdelta. In addition, PKCdelta and ERK1/2 were hyperphosphorylated in skin fibroblasts of CPEO patients harboring 4977 bp-deleted mtDNA. We suggest that the activation of PKCdelta and ERK1/2 elicited by 4977 bp-deleted mtDNA-induced oxidative stress plays a role in the susceptibility of the mutant cells to apoptosis. This may explain, at least in part, the degenerative manifestation of brain and muscle in patients with mitochondrial encephalomyopathies such as CPEO syndrome.

Preliminary investigation of greenhouse gas emissions from the environmental sector in Taiwan.

The United Nations Framework Conventions on Climate Change (UNFCCC) asks their Parties to submit a National Inventory Report (NIR) for greenhouse gas (GHG) emissions on an annual basis. However, when many countries are quickly growing their economy, resulting in substantial GHG emissions, their inventory reporting systems either have not been established or been able to be linked to planning of mitigation measures at national administration levels. The present research was aimed to quantify the GHG emissions from an environmental sector in Taiwan and also to establish a linkage between the developed inventories and development of mitigation plans. The "environmental sector" consists of public service under jurisdiction of the Taiwan Environmental Protection Administration: landfilling, composting, waste transportation, wastewater treatment, night soil treatment, and solid waste incineration. The preliminary results were compared with that of the United States, Germany, Japan, United Kingdom, and Korea, considering the gaps in the scopes of the sectors. The GHG emissions from the Taiwanese environmental sector were mostly estimated by following the default methodology in the Intergovernmental Panel on Climate Change guideline, except that of night soil treatment and waste transportation that were modified or newly developed. The GHG emissions from the environmental sectors in 2004 were 10,225 kilotons of CO2 equivalent (kt CO2 Eq.). Landfilling (48.86%), solid waste incineration (27%), and wastewater treatment (21.5%) were the major contributors. Methane was the most significant GHG (70.6%), followed by carbon dioxide (27.8%) and nitrous oxide (1.6%). In summary, the GHG emissions estimated for the environmental sector in Taiwan provided reasonable preliminary results that were consistent and comparable with the existing authorized data. On the basis of the inventory results and the comparisons with the other countries, recommendations of mitigation plans were made, including wastewater and solid waste recycling, methane recovery for energy, and waste reduction/sorting.

Enhanced x-ray irradiation-induced cancer cell damage by gold nanoparticles treated by a new synthesis method of polyethylene glycol modification.

We explored a very interesting gold nanoparticle system-pegylated gold in colloidal solution-and analyzed its uptake by mice colorectal adenocarcinoma CT26 tumor cells and the impact on the cell's response to x-ray irradiation. We found that exposure to polyethylene glycol (PEG) modified ('pegylated') 4.7 ± 2.6 nm gold nanoparticles synthesized by a novel synchrotron-based method enhances the response of CT26 cells to x-ray irradiation. Transmission electron microscopy (TEM) and confocal microscopy revealed that substantial amounts of such nanoparticles are taken up and absorbed by the cells and this conclusion is supported by quantitative induced coupled plasma (ICP) results. Standard tests indicated that the internalized particles are highly biocompatible but strongly enhance the cell damage induced by x-ray irradiation. Synchrotron radiation Fourier transform infrared (SR-FTIR) spectromicroscopy analyzed the chemical aspects of this phenomenon: the appearance of C = O stretching bond spectral features could be used as a marker for cell damage and confirmed the enhancement of the radiation-induced toxicity for cells.

Oxidative stress change by systemic corticosteroid treatment among patients having active graves ophthalmopathy.

OBJECTIVES: To measure the 8-hydroxy-2'-deoxyguanosine (8-OHdG) level in patients having active Graves ophthalmopathy (GO) and to compare this oxidative stress biomarker and the clinical evolution of patients after systemic corticosteroid treatment. METHODS: In 8 euthyroid patients having active GO, we determined the 8-OHdG levels in urine before, during, and after intensive corticosteroid therapy. Clinical activity and ophthalmopathy index scores were assessed. Nine age- and sex-matched healthy volunteers served as control subjects. RESULTS: The mean 8-OHdG level was statistically significantly increased in patients having active GO compared with that of controls (17.47 vs 5.97 ng/mg of creatinine, P < .001). During and after maximal systemic corticosteroid treatment, patients had statistically significantly lower mean 8-OHdG levels (7.19 and 10.18 ng/mg of creatinine, respectively) compared with the mean level before treatment. These changes were accompanied by decreases in clinical activity and ophthalmopathy index scores. The urinary 8-OHdG levels were subsequently elevated in 2 patients having recurrent active GO when corticosteroid therapy was tapered or withdrawn. CONCLUSIONS: Oxidative stress may have a role in the pathogenesis of GO. Urinary 8-OHdG level can be used not only as a noninvasive biomarker of oxidative stress in patients having GO but also as an objective and quantitative parameter in the follow-up of patients during immunosuppressive treatment.

Structural properties of naked gold nanoparticles formed by synchrotron X-ray irradiation.

The formation of colloidal unmodified (naked) gold nanoparticles is investigated by irradiation of a precursor solution with X-rays from a synchrotron source. An interesting morphological evolution as a function of exposure time, from cross-linked network-like structure to individual particles, has been discovered. The particle size decreased with the exposure time and was influenced by the ionic strength of the precursor solution. Contrary to gamma-ray exposure, an OH radical scavenger was not required for cluster formation.

A follow-up study in a Taiwanese family with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes syndrome.

BACKGROUND/PURPOSE: MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) syndrome is often associated with A3243G point mutation of mitochondrial DNA (mtDNA). We previously described a MELAS family characterized by harboring an additional approximately 260 bp tandem duplication in the D-loop and a novel C3093G point mutation in the 16S rRNA gene of mtDNA in the proband. We studied the clinical progression and fluctuation of mtDNA mutations in this Taiwanese MELAS family. METHODS: We followed up the clinical course in all members of this family (1 proband, her mother and 3 sons) for 12 years. Mutations of mtDNA in serial muscle biopsies of the proband and blood samples and hair follicles taken at different time points from the members of this family were analyzed. RESULTS: The proband developed repeated stroke-like episodes, chronic intestinal pseudo-obstruction, polyneuropathy, progressive renal failure and dilated cardiomyopathy with heart failure. During the follow-up period, the mother and one of the siblings of the proband developed stroke-like episodes at age 62 and 12, respectively. There was no significant difference in the proportions of mtDNA with A3243G mutation among five serial muscle biopsies of the proband. In one carrier (I-2), the proportion of A3243G mutated mtDNA in blood cells was slightly increased with disease progression. CONCLUSION: This study underlines the importance of early detection of extraneuromuscular symptoms in the members of a family with MELAS syndrome by adequate follow-up. The age of onset of stroke-like episode in MELAS syndrome may be as late as 62 years. We suggest that the manifestations of MELAS syndrome in this family might be associated with the additional approximately 260 bp tandem duplication in the D-loop region and the coexistence of C3093G mutation in the 16S rRNA gene with the A3243G mutation of mtDNA.

Quantitative effect of 4977 bp deletion of mitochondrial DNA on the susceptibility of human cells to UV-induced apoptosis.

In this study, we used a series of human cytoplasmic hybrids (cybrids) harboring different proportions of 4977 bp-deleted mtDNA to investigate the quantitative effect of a pathogenic mutation of mtDNA on apoptosis. We found that the sensitivity of human cells to apoptosis triggered by UV irradiation increases with the proportion of 4977 bp-deleted mtDNA. Moreover, UV-induced activation of caspase 3 was preceded by the activation of caspases 8 and 9. Most importantly, we observed that UV-induced cytochrome c release from mitochondria occurred much earlier and was much more pronounced in the cybrids harboring higher proportions of 4977 bp-deleted mtDNA. We suggest that 4977 bp-deleted mtDNA increases the susceptibility of human cells to UV-induced apoptosis in a quantitative manner through cytochrome c release from mitochondria and caspase 3 activation.

A common mitochondrial DNA variant and increased body mass index as associated factors for development of type 2 diabetes: Additive effects of genetic and environmental factors.

OBJECTIVE: The suggested correlation between a T-to-C transition at the nucleotide 16189 in mitochondrial DNA (mtDNA) with increasing insulin resistance and adult-onset diabetes mellitus (DM) is debatable. METHODS: Our study examined mtDNA from 462 subjects with type 2 diabetes (T2DM) and 592 normoglycemic controls (non-DM). Each participant's body mass index (BMI), fasting plasma glucose, fasting insulin concentration, insulin resistance index, and beta-cell function were measured. Sequencing for mtDNA, focusing on exploration of the hypervariable polycytosine tract within the control region, was also conducted in all subjects. RESULTS: Prevalence of the mtDNA 16189 variant was significantly different between DM and non-DM subjects (39.2% vs. 30.7% respectively; P = 0.004). Increased incidence of DM was noted in those harboring the 16189 variant compared with those lacking the variant (multivariate odds ratio, 1.38; 95% confidence interval, 1.07-1.80). Moreover, increased BMI was identified as an aggravating factor for development of DM in subjects harboring the variant. Odds ratio determinations yielded 2.14 in overweight and 4.63 in obese subjects harboring the variant in comparison with subjects without (1.83 in overweight and 2.16 in obese subjects). This is consistent with a progressively increased prevalence of the mtDNA 16189 variant in the non-DM groups with higher fasting insulin concentration, insulin resistance index, and beta-cell function (all P(trend) < 0.005). CONCLUSION: The mtDNA 16189 variant can influence development of T2DM. The demonstrated dynamic between the 16189 variant and increased BMI exemplify an additive effect of genetic and environmental factors on the pathogenesis of T2DM.

Involvement of protein kinase C delta in the alteration of mitochondrial mass in human cells under oxidative stress.

Alteration of mitochondrial mass of human 143B osteosarcoma cells upon exposure to hydrogen peroxide (H(2)O(2)) was investigated. We found that mitochondrial mass and the intracellular level of H(2)O(2) were increased by exogenous H(2)O(2), which was accompanied with up-regulation of functional PKCdelta. To investigate the role of PKCdelta in H(2)O(2)-induced increase of mitochondrial mass, we treated 143B cells with PKCdelta activator, bistratene A, and PKCdelta inhibitor, rottlerin, respectively. The results show that bistratene A caused an increase of mitochondrial mass and that the H(2)O(2)-induced increase of mitochondrial mass was completely suppressed by rottlerin. Furthermore, we found that activation of PKCdelta by bistratene A increased the intracellular levels of H(2)O(2) and MnSOD protein expression. By contrast, suppression of PKCdelta by rottlerin decreased the intracellular levels of H(2)O(2) and MnSOD protein expression. Moreover, we noted that MnSOD expression was highly correlated with the expression of p53, which was controlled by PKCdelta. Finally, we demonstrated that PKCdelta was overexpressed in skin fibroblasts of patients with MERRF syndrome. Taken together, we conclude that PKCdelta is involved in the regulation of mitochondrial mass and intracellular H(2)O(2) in human cells and may play a key role in the overproliferation of mitochondria in the affected tissues of patients with mitochondrial diseases such as MERRF syndrome.

Accumulation of mitochondrial DNA with 4977-bp deletion in knee cartilage--an association with idiopathic osteoarthritis.

OBJECTIVE: Since mitochondrial DNA (mtDNA) mutations have been established to associate with the aging process and some degenerative diseases, we investigated the correlation between idiopathic osteoarthritis (OA) and the 4977-bp mtDNA deletion. DESIGN: Cartilage were collected from six sites in knee joints removed from 18 aged patients with idiopathic OA, 10 aged non-OA cadavers, 3 young cadavers (YC), and lateral femoral condyle of 9 young patients. Histopathologic changes were examined and the common 4977-bp mtDNA deletions were analyzed in young and elderly cartilages obtained from different sites in the knee joint. The association of the 4977-bp deletion of mtDNA with idiopathic OA and aging was evaluated. RESULTS: The 4977-bp mtDNA deletion was detected in 17 of the 18 OA patients, 9 of the 10 aged non-OA cadavers, and 1 of the 3 YC. None of the nine specimens collected from the lateral femoral condyle of young patients had a detectable deletion of mtDNA. The 4977-bp mtDNA deletion was not significantly correlated with the severity of OA graded by the Mankin score. The frequencies of occurrence of the 4977-bp mtDNA deletion were significantly different between the OA group and the aged non-OA control group (P=0.004) and between the aged non-OA group and the young control group (P=0.002). CONCLUSIONS: The results suggest that accumulation of the 4977-bp deletion of mtDNA in knee cartilage increases with age and may play a role in the development of idiopathic OA in the knee joint.

Association of mitochondrial deoxyribonucleic acid 16189 variant (T->C transition) with metabolic syndrome in Chinese adults.

OBJECTIVE: A common variant in mitochondrial DNA (mtDNA) at bp 16189 (T-->C transition) has been associated with small birth size, adulthood hyperglycemia, and insulin resistance in Caucasians. In this study, we investigated whether mtDNA 16189 variant is associated with metabolic syndrome in Chinese subjects. METHODS: Six hundred fifteen Chinese adults, aged 40 yr or older, were recruited in this study. The 16189 variant of mtDNA was detected using PCR and restriction enzyme digestion. Metabolic syndrome was diagnosed on modified National Cholesterol Education Program Adult Treatment Panel III guidelines, using body mass index (BMI) instead of waist circumference. An association study was performed with chi2 test and logistic regression analysis. RESULTS: The prevalence of the 16189 variant was higher in patients with metabolic syndrome than in those without: 44% (125 of 284) vs. 33.2% (110 of 331) (P = 0.006). The association between this 16189 variant of mtDNA and metabolic syndrome (P = 0.021) remained significant even after correcting for age and BMI. As to the individual traits, the prevalence of fasting plasma glucose of at least 110 mg/dl (> or =6.1 mmol/liter) [(51.5% (121 of 235) vs. 42.1% (160 of 380); P = 0.023], type 2 diabetes mellitus [48.1% (113 of 235) vs. 39.2% (149 of 380); P = 0.031], and hypertriglyceridemia [44.3% (104 of 235) vs. 35.8% (136 of 380); P = 0.037] were significantly higher in subjects harboring the 16189 variant of mtDNA than those with the wild type. However, the prevalence of hypertension [53.2% (125 of 235) vs. 47.6% (181 of 380); P = 0.180], BMI greater than 25 kg/m2 [48.5% (114 of 235) vs. 43.9% (167 of 380); P = 0.270], and low high-density lipoprotein cholesterol [61.3% (144 of 235) vs. 54.7% (208 of 380); P = 0.111] did not reach a significant difference between the two groups. Furthermore, there was a trend of increasing frequency of occurrence of the 16189 variant in individuals having an increasing number of components of metabolic syndrome (Ptrend < 0.005). CONCLUSION: Our data strongly suggest that mtDNA 16189 variant underlies susceptibility to metabolic syndrome in the Chinese population.

Alteration of the copy number of mitochondrial DNA in leukocytes of patients with hyperlipidemia.

Lipid metabolism in leukocytes may be disturbed by mitochondrial dysfunction caused by depletion of mitochondrial DNA (mtDNA) in response to an increase of oxidative stress in blood circulation. It is possible that alteration in mtDNA copy number of the leukocyte is involved in the impairment of the scavenging of oxidatively modified plasma proteins such as oxidized low-density lipoprotein (oxLDL). To test this hypothesis, we recruited 91 healthy subjects and 63 patients with hyperlipidemia (LDL >130 mg/dL) for this study. The copy number of mtDNA in the leukocyte and the titer of oxLDL IgG autoantibody (oLAB) were determined as indices of the oxidative stress response of immune cells. The results revealed a significant higher level of plasma oxLDL, lower titer of oLAB, and decreased copy number of mtDNA in patients with hyperlipidemia (P <0.05). In the analysis of partial correlations under age control, we found that an increase in the copy number of mtDNA was positively correlated with an increase in the level of oLAB (P <0.005, r = 0.3002) and a decrease in the oxLDL level (P <0.05, r = -0.2654) in healthy subjects but not in patients. Based on the results obtained from this case-control study, we conclude that the increase of mtDNA copy number might provide the leukocyte an increased capability of scavenging oxLDL, possibly by enhanced generation of oLAB in healthy subjects, but not in hyperlipidemic patients who had lower mtDNA copy numbers in their leukocytes. Taken together, these findings suggest that an alteration of mtDNA copy number in the leukocyte may be one of the risk factors for hyperlipidemia.

Increased oxidative damage with altered antioxidative status in type 2 diabetic patients harboring the 16189 T to C variant of mitochondrial DNA.

A transition of T to C at nucleotide position 16189 in mitochondrial DNA (mtDNA) has attracted biomedical researchers for its probable correlation with the development of diabetes mellitus in adult life. In diabetes, persistent hyperglycemia may cause high production of free radicals. Reactive oxygen species are thought to play a role in a variety of physiologic and pathophysiologic processes in which increased oxidative stress may play an important role in disease mechanisms. The aim of the present study was to clarify the degree of oxidative damage and plasma antioxidant status in diabetic patients and to see the potential influence of the 16189 variant of mtDNA on the oxidative status in these patients. An indicative parameter of lipid peroxidation, malondialdehyde (MDA), and total free thiols were measured from plasma samples of 165 type 2 diabetic patients with or without this variant and 168 normal subjects. Here we report an increase in the plasma levels of MDA and total thiols in type 2 diabetic patients compared with control subjects. The levels of plasma thiols in diabetic patients with the 16189 variant of mtDNA were not different from those in controls. These results suggest an increase in the oxidative damage and a compensatory higher antioxidative status in patients with type 2 diabetes. Harboring the 16189 mtDNA variant may impair the ability of a cell to respond properly to oxidative stress and oxidative damage.

Oxidative stress-induced depolymerization of microtubules and alteration of mitochondrial mass in human cells.

Mitochondrial biogenesis is a biological process that has been intensively studied over the past few years. However, the detailed molecular mechanism underlying this increase in mitochondria remains unclear. To investigate the mechanism of such a mitochondrial proliferation, we examined alterations in mitochondria of human osteosarcoma 143B cells that had been treated with 100 to 500 microM hydrogen peroxide (H2O2) for 48 h. The results showed that mitochondrial mass of the cell was increased with the increase of the concentration of H2O2. On the other hand, by using real-time PCR techniques, we observed the changes of mitochondrial DNA (mtDNA) content in the cells exposed to oxidative stress. The copy number of mtDNA was increased by treatment with a low dose of H2O2 but was drastically decreased after treatment with H2O2 higher than 300 microM. Transmission electron microscopic images revealed that mitochondria were abnormally proliferated in cells exposed to oxidative stress. Moreover, we found that the percentage of 143B cells arrested at the G2/M phase increased upon treatment with H2O2. Immunostaining and microtubule fractionation assay revealed that microtubules were depolymerized in the cells that had been treated with H2O2. To understand the effect of microtubules depolymerization on the mitochondrial mass, we treated the cells with several kinds of microtubule-active drugs, which arrest cultured cells at the G2/M phase. The results showed that mitochondrial mass and mtDNA copy number all were increased after such treatments. Taking these findings together, we suggest that oxidative stress-induced microtubule derangement is one of the molecular events involved in the increase of mitochondrial mass upon treatment of human cells with H2O2.

Attenuation of UV-induced apoptosis by coenzyme Q10 in human cells harboring large-scale deletion of mitochondrial DNA.

Chronic progressive external ophthalmoplegia (CPEO) syndrome is one of the mitochondrial diseases caused by large-scale deletions in mitochondrial DNA (mtDNA) that impair the respiratory function of mitochondria and result in decreased production of ATP in affected tissues. In order to investigate whether CPEO-associated mtDNA mutations (i.e., 4,366-bp and 4,977-bp large-scale deletions) render human cells more vulnerable to apoptosis, we constructed cybrids carrying the deleted mtDNA. Assays for cell viability, DNA fragmentation, cytochrome c release, and caspase 3 activation revealed that UV irradiation at 20 J/m2 triggered apoptosis in all the cybrids. This treatment also produced elevated intracellular levels of reactive oxygen species (ROS). The rate of UV-induced cell death was more pronounced in the cybrids harboring mtDNA deletions than in the control cybrid with wild-type mtDNA. Subsequently, we evaluated the effect of coenzyme Q10 on the UV-triggered apoptosis. The results showed that after pretreatment of the cybrids with 100 microM coenzyme Q10 the UV-induced cell damage (i.e., ROS production and activation of caspase 3) was significantly reduced. Taken together, these findings suggest that large-scale deletions of mtDNA increased the susceptibility of human cells to the UV-triggered apoptosis and that coenzyme Q10 mitigated the damage; hence, it might potentially serve as a therapeutic agent to treat mitochondrial diseases resulting from mtDNA deletions.