Effects of (-)-sesamin on motor and memory deficits in an MPTP-lesioned mouse model of Parkinson's disease treated with l-DOPA.

The present study investigated the effects of (-)-sesamin on motor and memory deficits in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of Parkinson's disease (PD) with l-3,4-dihydroxyphenylalanine (l-DOPA). MPTP-lesioned (30mg/kg/day, 5days) mice showed deficits in memory including habit learning memory and spatial memory, which were further aggravated by daily treatment with 25mg/kg l-DOPA for 21days. However, daily treatment with (-)-sesamin (25 and 50mg/kg) for 21days ameliorated memory deficits in an MPTP-lesioned mouse model of PD treated with l-DOPA (25mg/kg). Both (-)-sesamin doses reduced decreases in the retention latency time in the passive avoidance test, latency to fall of rotarod test and distance traveled in the open field test, and attenuated decreases in tyrosine hydroxylase (TH)-immunopositive cells, dopamine, and its metabolites in the substantia nigra-striatum. (-)-Sesamin reduced increases in the retention transfer latency time in the elevated plus-maze test and N-methyl-d-aspartate receptor (NMDAR) expression and reduced decreases in the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-response element binding protein (CREB) in the hippocampus. In contrast, daily treatment with 10mg/kg l-DOPA for 21days ameliorated memory deficits in MPTP-lesioned mice, and this effect was further improved by treatment with (-)-sesamin (25 and 50mg/kg). These results suggest that (-)-sesamin protects against habit learning memory deficits by activating the dopamine neuronal system, while spatial memory deficits are decreased by its modulatory effects on the NMDAR-ERK1/2-CREB system. Accordingly, (-)-sesamin may act as an adjuvant phytonutrient for motor and memory deficits in patients with PD receiving l-DOPA.

Exopolysaccharide fraction from Pediococcus pentosaceus KFT18 induces immunostimulatory activity in macrophages and immunosuppressed mice.

AIMS: Exopolysaccharide fraction from Pediococcus pentosaceus KFT18 (PE-EPS), a lactic acid bacteria isolated from Kimchi (a Korean fermented vegetable product), was preliminary characterized and its immunostimulating effects were analysed. METHODS AND RESULTS: In this study, we used interferon-γ (IFN-γ)-primed RAW 264·7 macrophages and CD3/CD28-stimulated splenocytes to determine the immunotimulatory activities of PE-EPS. Upon exposure to PE-EPS, IFN-γ-primed RAW 264·7 macrophages showed significant increases in the expressions of inducible nitric oxide synthase (iNOS), tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß. Molecular data using reporter gene assay and electrophoretic mobility shift assay (EMSA) revealed that PE-EPS upregulated transcriptional activity, DNA binding and the nuclear translocation of nuclear factor-κB (NF-κB). Furthermore, PE-EPS enhanced anti-CD3/CD28-specific proliferation and the productions of IL-2 and IFN-γ in primary splenocytes. In cyclophosphamide-induced immunosuppressed mice, pretreatment with PE-EPS (5, 15 or 45 mg kg(-1) day(-1), p.o.) increased thymus and spleen indices, and improved lymphocyte and neutrophil counts. CONCLUSION: PE-EPS stimulated the IFN-γ-primed macrophages and primary splenocytes to induce immune responses and improved the cyclophosphamide-induced immunosuppression in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: The results in this study improved our understanding of immunostimulating activity of PE-EPS and supported its potential treatment option as a natural immunostimulant.

Polysaccharides from Korean Citrus hallabong peels inhibit angiogenesis and breast cancer cell migration.

Although the peel of the hallabong (Citrus sphaerocarpa) fruit is rich in polysaccharides, which are valuable dietary ingredients for human health, it is normally wasted. The present study aimed to utilize the peel waste and identify properties it may have against breast cancer metastasis. Hallabong peel extract containing crude polysaccharides was fractionated by gel permeation chromatography to produce four different polysaccharide fractions (HBE-I, -II, -III, and -IV). The HBE polysaccharides significantly blocked tube formation of human umbilical vein vascular endothelial cells (HUVECs), at a concentration of 12.5 or 25 µg/mL. Tube formation appeared to be more sensitive to HBE-II than to other HBE polysaccharides. HBE-II also inhibited breast cancer cell migration, through downregulation of matrix metalloproteinase-9 (MMP-9) in MDA-MB-231 triple-negative breast cancer cells. Therefore, inhibition of tube formation and MMP-9-mediated migration observed in HUVEC and MDA-MB-231 cells, respectively, are likely to be important therapeutic targets in triple-negative breast cancer metastasis.

An epidemiological study of androgenic alopecia in 3114 Korean patients.

BACKGROUND: Androgenetic alopecia (AGA) is the most common type of hair loss, and is characterized by the transformation of terminal scalp hair into vellus hair. The epidemiology of AGA is not fully understood. A strong genetic basis has long been identified, although little is known of its nongenetic causes. AIM: To evaluate the association of AGA with a number of environmental factors, including smoking, drinking and sleeping habit. METHODS: In total, 3114 Korean individuals with AGA who attended any one of 17 dermatology clinics in 6 cities in South Korea between March 2011 and February 2012 were enrolled in the study. Epidemiologic a data were collected using a standard questionnaire. RESULTS: No association was seen between eating or sleeping habits and severity of hair loss. However, drinking and smoking were associated with the severity of AGA in male patients. We also found that patients of both genders with a family history had more advanced types of hair loss, and the age of onset of AGA in male patients with a family history was earlier than that in male patients without a family history. CONCLUSIONS: Although the evidence for an environmental influence on AGA remains very weak, we did find an association between hair loss severity and certain environmental factors, such as smoking and drinking. Family history with more severe hair loss and an earlier age of onset.

CHIP has a protective role against oxidative stress-induced cell death through specific regulation of endonuclease G.

Oxidative stress is implicated in carcinogenesis, aging, and neurodegenerative diseases. The E3 ligase C terminus of Hsc-70 interacting protein (CHIP) has a protective role against various stresses by targeting damaged proteins for proteasomal degradation, and thus maintains protein quality control. However, the detailed mechanism by which CHIP protects cells from oxidative stress has not been demonstrated. Here, we show that depletion of CHIP led to elevated Endonuclease G (EndoG) levels and enhanced cell death upon oxidative stress. In contrast, CHIP overexpression reduced EndoG levels, and resulted in reduced or no oxidative stress-induced cell death in cancer cells and primary rat cortical neurons. Under normal conditions Hsp70 mediated the interaction between EndoG and CHIP, downregulating EndoG levels in a Hsp70/proteasome-dependent manner. However, under oxidative stress Hsp70 no longer interacted with EndoG, and the stabilized EndoG translocated to the nucleus and degraded chromosomal DNA. Our data suggest that regulation of the level of EndoG by CHIP in normal conditions may determine the sensitivity to cell death upon oxidative stress. Indeed, injection of H2O2 into the rat brain markedly increased cell death in aged mice compared with young mice, which correlated with elevated levels of EndoG and concurrent downregulation of CHIP in aged mice. Taken together, our findings demonstrate a novel protective mechanism of CHIP against oxidative stress through regulation of EndoG, and provide an opportunity to modulate oxidative stress-induced cell death in cancer and aging.

Gigacycle fatigue behavior by ultrasonic nanocrystalline surface modification.

Nanocrystalline surface layer up to 84 microm in thick is produced on a specimen made of Al6061-T6 alloy by means of surface treatment called ultrasonic nanocrystalline surface modification (UNSM) technique. The refined grain size is produced in the top-layer and it is increased with increasing depth from the top surface. Vickers microhardness measurement for each nanocrystalline surface layer is performed and measurement results showed that the microhardness is increased from 116 HV up to 150 HV, respectively. In this study, fatigue behavior of Al6061-T6 alloy was studied up to 10(7)-10(9) cycles by using a newly developed ultrasonic fatigue testing (UFT) rig. The fatigue results of the UNSM-treated Al6061-T6 alloy specimens were compared with those of the untreated specimens. The microstructure of the untreated and UNSM-treated specimens was characterized by means of scanning electron microscopey (SEM) and transmission electron microscopey (TEM).

Nonstructural 5A protein of hepatitis C virus regulates heat shock protein 72 for its own propagation.

We identified heat shock protein 72 (Hsp72) as a host factor that was differentially expressed in cells expressing nonstructural 5A (NS5A) protein. To investigate how NS5A modulates Hsp72 in hepatitis C virus (HCV) life cycle, we examined the role of Hsp72 in HCV replication and virus production. NS5A specifically interacted with Hsp72. Both Hsp72 and nuclear factor of activated T cells 5 (NFAT5) levels were increased in cells expressing NS5A protein. Treatments of N-acetylcysteine and glutathione markedly reduced protein levels of both NFAT5 and Hsp72. Knockdown of NFAT5 resulted in decrease in Hsp72 level in cells expressing NS5A. Importantly, silencing of Hsp72 expression resulted in decrease in both RNA replication and virus production in HCV-infected cells. These data indicate that NS5A modulates Hsp72 via NFAT5 and reactive oxygen species activation for HCV propagation.

The role of sirtuin 1 in osteoblastic differentiation in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Activation of sirtuin 1 (SIRT1) promotes the differentiation of keratinocytes and mesenchymal stem cells, but inhibits the differentiation of muscle and fat cells. However, the involvement of SIRT1 in the differentiation of human periodontal ligament cells into osteoblast-like cells remains unclear. To identify the role of SIRT1 in human periodontal ligament cells, we measured SIRT1 mRNA and SIRT1 protein levels during the osteoblastic differentiation of human periodontal ligament cells. Additionally, we investigated the effects of overexpressing and underexpressing SIRT1 on the differentiation of human periodontal ligament cells, and the signaling mechanisms involved. MATERIAL AND METHODS: Expression of SIRT1 and osteoblastic differentiation markers was assessed by RT-PCR, real-time PCR, Alizarin red staining and western blotting. RESULTS: Marked upregulation of SIRT1 mRNA and SIRT1 protein was observed in cells grown for 3 d in osteogenic induction medium (OM). Activation of SIRT1 using resveratrol and isonicotinamide stimulated osteoblastic differentiation in a dose-dependent manner, as assessed by the expression of mRNAs encoding alkaline phosphatase, osteopontin, osteocalcin, osterix and Runx2, and induced calcium deposition. In contrast, inhibition of SIRT1 using sirtinol, nicotinamide and gene silencing by RNA interference suppressed mineralization and the expression of osteoblast marker mRNAs. Further mechanistic studies revealed that resveratrol treatment increased the phosphorylation of Akt, adenosine monophosphate kinase (AMPK), Smad 1/5/8 and c-Jun N-terminal kinase, but reduced OM-induced activation of nuclear factor-κB. Conversely, application of sirtinol suppressed the phosphorylation of Akt, AMPK, Smad 1/5/8, p38, ERK and c-Jun N-terminal kinase, and enhanced nuclear factor-κB activity, in OM-stimulated cells. CONCLUSION: These data suggest that SIRT1 is a potent regulator of differentiation of human periodontal ligament cells and may have clinical implications for periodontal bone regeneration.

Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serum.

OBJECTIVES: Translational research using adult stem cells derived from various tissues has been highlighted in cell-based therapy. However, there are many limitations to using conventional culture systems of adult stem cells for clinically applicability, including limited combinations of cytokines and use of nutrients derived from animals. Here, we have investigated the effects of placental extract (PE) for culture of placenta-derived stem cells (PDSCs) as well as their potential for hepatogenic differentiation. MATERIALS AND METHODS: Placental extract, extracted using water-soluble methods, was used as a supplement for culture of PDSCs. Cell viability was determined using the MTT assay, and cytokine assay was performed using Luminex assay kit. Gene expression, indocyanine green (ICG) up-take, PAS (Periodic Acid-Schiff) staining and urea production were also analysed. RESULTS: The placental extract contained several types of cytokine and chemokine essential for maintenance and differentiation of stem cells. Expression of stemness markers in PDSCs cultured with PE is no different from that of PDSCs cultured with foetal bovine serum (FBS). After hepatogenic differentiation, expression patterns for hepatocyte-specific markers in PDSCs cultured with PE were consistent and potential for hepatogenic differentiation of PDSCs cultured with PE was similar to that of PDSCs cultured with FBS, as shown by PAS staining and urea production assays. CONCLUSIONS: Our findings revealed that placental extract could be used as a new component for culture of adult stem cells, as well as for development of human-based medium, in translational research for regenerative medicine.

The role of psychological distress in laryngopharyngeal reflux patients: a prospective questionnaire study.

OBJECTIVES: To determine the role of psychological distress in laryngopharyngeal reflux patients and evaluate the correlation between symptoms, laryngeal signs, pH monitoring results and psychological profile. DESIGN: Prospective study. SETTING: Hanyang University Hospital, a university teaching hospital and tertiary referral center. PARTICIPANTS: One hundred and six patients who were diagnosed with laryngopharyngeal reflux by 24-h ambulatory double probe pH monitoring and 119 healthy controls visiting our health promotion center from January 2006 to June 2007. MAIN OUTCOME MEASURES: The psychological profile of laryngopharyngeal reflux patients measured by the Symptom Checklist-90-Revised questionnaire were evaluated and compared with those of healthy controls. The correlation between reflux symptom index, reflux finding score, parameters of pH monitoring and the Symptom Checklist-90-Revised profiles were also evaluated. RESULTS: On the Symptom Checklist-90-Revised questionnaire, the total mean T-scores of the nine symptom dimensions and three global indices of the laryngopharyngeal reflux patients were all below 50. The Global Severity Index, which indicates overall psychological distress, was normal in all of the patients. On comparison with the control group, no statistically significant difference was noted in the psychological profile except on the Somatisation scale where laryngopharyngeal reflux patients showed significantly higher scores. Reflux symptom index showed significant positive correlation with the number of reflux episodes, percentage of time which pH fell below 4 in total positions, and DeMeester score of the upper probe. The nine symptom dimensions and three global indices of Symptom Checklist-90-Revised questionnaire did not show any correlation with reflux symptom index, reflux finding score and the parameters of the 24-h ambulatory double probe pH monitoring. CONCLUSIONS: Laryngopharyngeal reflux patients did not demonstrate any significant level of psychological distress and their symptom severity showed significant positive correlation with reflux severity.

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production.

AIMS: Elucidation of the regulation of ChiB production in Aspergillus nidulans. METHODS AND RESULTS: Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures. CONCLUSIONS: The endochitinase ChiB plays an important role in autolysis of A. nidulans, and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling. SIGNIFICANCE AND IMPACT OF THE STUDY: Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.

CT colonography in a Korean population with a high residue diet: comparison between wet and dry preparations.

AIM: To compare wet and dry preparation methods for computed tomography colonography (CTC) in terms of preparation quality, interpretation time, and diagnostic performance for polyp detection in a population with a high residue diet. MATERIALS AND METHODS: Eighty-six patients were divided into two groups. Group 1 (n=24) received a wet preparation of 4l polyethylene glycol (PEG) solution, and group 2 (n=62) received a dry preparation of phosphor-soda. Abnormal findings, including polyps, and the time required to interpret the CTC images in both groups were documented by a radiologist. CTC findings were compared to those of colonoscopy as a reference standard. Two radiologists evaluated the quality of CTC with regard to residual fluid, faeces, and colonic distension using a four-point scale in consensus. Statistical differences for residual fluid, faeces, distensibility on CTC, and interpretation time between the two groups were analysed. The diagnostic performance of CTC in both groups was also compared. RESULTS: One-hundred and ninety polyps in 70 patients were identified using colonoscopy. Regarding the quality of images produced the wet preparation was significantly better than the dry preparation (p<0.05). The average interpretation time was significantly shorter for the wet group (11.7 min) than the dry group (16.4 min) (p<0.05). For per-patient analysis, the positive predictive value (PPV) was significantly better for the wet (100%) than the dry group (79.6%; p=0.025). Sensitivities and PPV for >or=10 mm polyps were comparable between two groups (p>0.05). CONCLUSION: In a population with a high-residue diet, CTC with wet preparation can be interpreted in a time-efficient manner and is comparable with CTC with dry preparation.

Hyperhydricity in apple: ultrastructural and physiological aspects.

We studied the effects of hyperhydricity on subcellular ultrastructure and physiology of leaves during in vitro regeneration of apple plants. Morphological, anatomical and ultrastructural differences between healthy leaf tissues obtained from greenhouse-grown plants and healthy and hyperhydric leaves obtained from shoots raised from nodal shoot explants in a bioreactor were investigated by electron microscopy and confocal laser scanning microscopy. Compared with healthy leaves, hyperhydric leaves showed abnormal, often discontinuous development of the epidermis and cuticle. Stomata were malformed. The leaf lamina appeared thickened and was characterized by poor differentiation between the palisade and spongy mesophyll tissue. Hyperhydric leaves had a significantly lower chloroplast number per cell and chloroplasts showed reduced thylakoid stacking compared with healthy leaves. Hyperhydricity resulted in a general decrease in concentrations of reduced and oxidized pyridine nucleotides, reflecting a reduction in metabolic activity. The activities of antioxidant enzymes, such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase were higher in hyperhydric leaves than in healthy leaves, indicating that hyperhydricity was associated with oxidative stress. Chlorophyll fluorescence measurements provided evidence of oxidative damage to the photosynthetic machinery in hyperhydric leaves: photochemical efficiency of photosystem II, effective quantum efficiency and photochemical quenching were all lower in hyperhydric leaves compared with healthy leaves.

An ex-vivo experimental study on optimization of bipolar radiofrequency liver ablation using perfusion-cooled electrodes.

PURPOSE: To determine optimal parameters for bipolar radiofrequency ablation (RFA) using perfusion-cooled electrodes to create a large ablation volume in ex vivo bovine liver. MATERIAL AND METHODS: Three sets of RF experiments were performed using a 200-Watt generator and two 15-gauge perfusion-cooled or internally cooled electrodes in ex vivo bovine livers. In the first set of experiments, to find the ideal inter-electrode distance for creating large coagulation necrosis, 30 ablation lesions were created by bipolar RFAs at inter-electrode spacings of 3 cm, 4 cm, and 5 cm. In the second set of experiments, to explore the ideal duration of RF application, bipolar RFAs were performed for 10 min and 20 min. In the first and second experiments, 10 lesions were made for each condition with infusion of 6% hypertonic saline (HS) at 2 ml/min. In the third set of experiments, 10 ablation lesions were created by bipolar RFAs using internally cooled electrodes without HS infusion. The mean volume of those ablation lesions was then compared to that of the lesions created by bipolar RFA using perfusion-cooled electrodes in the second experiments. Tissue impedance, dimension, and shape of the ablated areas were compared in each condition. RESULTS: In the first set of experiments, bipolar RFA created a homogeneous oval or spherical-shaped ablation area between the electrodes at 3-5 cm spacing, but showed a more spherical-shaped lesion at 3 cm inter-electrode spacing than at 4 cm and 5 cm spacing. In the second set of experiments, RF energy delivered for 20 min created a larger dimension of coagulation necrosis than energy delivered for 10 min: 107.6 +/- 34 cm3 versus 59.5 +/- 27 cm3 (P<0.05). In addition, the mean volume of ablation regions obtained with bipolar RFA using the internally cooled electrode was 47.5+/- 17 cm3, which was significantly less than that with bipolar RFA using perfusion-cooled electrodes (P <0.05). CONCLUSION: Bipolar RFA using perfusion-cooled electrodes achieves homogeneous areas of coagulation necrosis between two electrodes, preferably at 3 or 4 cm inter-electrode distance for 20 min, and is better in creating large coagulation necrosis than bipolar RFA using internally cooled electrodes.

Macrophage-stimulating activity of exo-biopolymer from cultured rice bran with Monascus pilosus.

To find a new use of rice bran, five fungi were examined for the production of exo-biopolymer with macrophage-stimulating activity from rice bran. Among the exo-biopolymers produced from the cultures, Monascus pilosus had the most potent macrophage stimulating activity in a liquid culture rather than in a solid culture. In order to improve the yield of exo-biopolymer with macrophage-stimulating activity, a suitable medium for exo-biopolymer was tested in submerged culture of M. pilosus. The highest amount of exo-biopolymer (13.9 mg/mL) was obtained in a medium containing rice bran as an only carbon source followed by media with additional maltose and sucrose (13.8 and 13.7 mg/mL, respectively). The addition of peptone resulted in the production of high amount of exo-biopolymer (15.1 mg/mL), meanwhile the addition of ammonium chloride resulted in 264.0 microg/mL of glucosamine content. Among eight different kinds of inorganic salts tested, potassium phosphate (0.1%) was the most effective inorganic salt for the mycelial growth and exo-biopolymer production. Therefore the optimal medium composition was as follows (g/L): 20 g of rice bran, 5 g of peptone, and 1 g of KH2PO4. The optimal culture pH and time for mycelial growth and exo-biopolymer production was pH 5.0 and 25 degrees C, respectively. The maximum exo-biopolymer (20.1 mg/mL) was observed at the fourth day of cultivation. Exo-biopolymer, a crude polysaccharide fraction, mainly contained neutral sugar (81.8%) with considerable amounts of uronic acid (18.2%). Component sugar analysis showed that the active fraction consisted mainly of arabinose, galactose, glucose, which was digested from starch of rice bran during cultivation, and uronic acid (molar ratio; 0.8:1.0:0.7:0.8).

Association of the oestrogen receptor alpha gene polymorphisms with disease onset in systemic lupus erythematosus.

OBJECTIVE: To evaluate the genetic influence of PvuII and XbaI polymorphisms of oestrogen receptor alpha (ORalpha) in patients with systemic lupus erythematosus (SLE) in Korea. METHODS: Genomic DNA from 268 female controls and 137 female SLE patients (41 childhood onset and 96 adult onset) were analysed using PvuII and XbaI restriction fragment length polymorphism. Comparison of the frequencies of alleles and genotypes was made in control and patient groups and in childhood onset and adult onset SLE subgroups. RESULTS: Although the Pp genotype occurred more often in SLE patients than in controls (p(c) = 0.017), ORalpha genotype distributions of adult onset SLE did not differ significantly from controls. The PP, Pp, and xx genotypes occurred less often in childhood onset SLE (p(c) = 0.0045, 0.0498, and 0.0255, respectively) than in controls. Additionally, the PP genotype was less common in childhood onset than in adult onset SLE (p(c) = 0.016). SLE patients with the PP genotypes were older at disease onset than those with the other genotypes (p = 0.001). Patients with the Xx genotype had an earlier onset of SLE than those with the xx genotype (p = 0.025). The frequency of the combined ppXx genotype was greater in childhood onset SLE than in controls (p(c) = 0.0009) or adult onset SLE (p(c) = 0.027). The same trend was supported by subgroup analyses according to age at menarche and logistic multivariate analyses. CONCLUSIONS: ORalpha polymorphisms are significantly associated with the age at disease onset in Koreans with SLE.

Alpha-difluoromethylornithine, ornithine decarboxylase inhibitor, antagonizes H2O2-induced cytotoxicity in HL-60 leukemia cells: regulation of iron-dependent lysosomal damage.

Recent studies indicate that reactive oxygen species, such as H2O2, can be generated by anti-cancer drugs, can damage cells, and then induce apoptotic cell death. In this study, we reported whether polyamines were capable of affecting apoptotic cell death triggered by H2O2 in leukemia cells or not. Alpha-difluoromethylornithine treatment (DFMO, 3 mmol/L, 48 h), which depletes intracellular putrescine by inhibiting ornithine decarboxylase, reduced H2O2-induced cell death in the HL-60 leukemia cells. Cytotoxicity caused by H2O2 in putrescine-depleted cells was 50% lower than that in the control cells, as determined by propidium iodide, the annexin V and DNA fragmentation assays. Following putrescine (1 mmol/L) supplement, cell death induction caused by H2O2 was restored to a similar level as the DFMO-untreated control cells. It seems that this partly resulted from the intralysosomal iron-dependent oxidation of the cells because DFMO did not significantly affect the increment of enzymes related to oxidative-stress resistance. Putrescine depletion by DFMO treatment reduced the cellular iron uptake of the cells by about 70%. In parallel to the reduction of iron uptake, lysosomal damage (assayed by acridine orange relocalization or uptake test) in the DFMO-treated cells was far less than that in the control cells. Moreover, putrescine supplement also restored the iron uptake to the control cell levels. Pre-incubation with desferrioxamine (DFO), which chelates iron and forms a non-reactive Fe-DFO complex that is localized in the lysosomal compartment, inhibited H2O2-induced cell death. This work suggests that polyamines may play a critical role in apoptotic cell death triggered by H2O2 via the regulation of the iron-dependent instability of the lysosome.

Phylogenetic relationships between the six superoxide dismutase proteins (FeSOD) of Trichomonas vaginalis and FeSOD6 genetic diversity.

The parasitic protozoan Trichomonas vaginalis is known to contain several types of Fe-containing superoxide dismutase proteins (FeSOD). Using three different methods of phylogenetic analysis, maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) methods, we examined the phylogenetic relationships among the six FeSOD (FeSOD1-FeSOD6) based on their amino acid sequences. All the analyses consistently suggested that the six proteins formed a monophyletic group implying that they probably be originated from an ancestral protein form through repeated duplication events. Although MP tree was totally unresolved, the NJ and ML trees revealed that FeSOD6 placed the most basal position and thus emerged earlier than the other five gene types during the evolution of T. vaginalis. Phylogenetic relationships among the five remaining proteins were (FeSOD2, FeSOD3), (FeSOD4, (FeSOD1, FeSOD5)) although weakly supported in terms of bootstrapping values. In addition to this, we newly designed two PCR primer specifically amplifying full-length FeSOD6 gene and examined its genetic diversity among 12 T. vaginalis isolates from five countries and three continents. They had the same nucleotide sequences except those of three Korean isolates which showed one to three different nucleotides.