A theoretical characterization method for non-spherical core-shell nanoparticles by XPS.

Core-shell nanoparticles (NPs) are active research areas for their unique properties and wide applications. By changing the elemental composition in the core and shell, a series of core-shell NPs with specific functions can be obtained, where the sizes of the core and shell also influence the properties. X-ray photoelectron spectroscopy (XPS) is useful in this context as a means of quantitatively analyzing such NPs. The empirical formula proposed by Shard [J. Phys. Chem. C, 2012, 116(31), 16806-16813] for calculating the shell thickness of the spherical core-shell NPs has been verified by Powell et al. [J. Phys. Chem. C, 2016, 120(39), 22730-22738] through a simulation of XPS with Simulation of Electron Spectra for Surface Analysis (SESSA) software. However, real core-shell NPs are not necessarily ideal spheres; such NPs can have rich shapes and uneven thicknesses. This work aims to extend the Shard formula to non-ideal core-shell NPs. We have used a Monte Carlo simulation method to study the XPS signal variation with the shell thickness for several modeled non-spherical shapes of core-shell NPs including some complex geometric structures which are numerically constructed with finite-element triangular meshes. Five types of non-spherical shapes, i.e. egg, ellipsoid, rod, rough-surface, and star shapes, are considered, while the size parameters are varied over a wide range. The equivalent radius and equivalent thickness are defined to characterize the average size of the nanoparticles for the use of the Shard formula. We have thus derived an extended Shard formula for the specific core-shell NPs, with which the relative error between the predicted shell thickness and the real thickness can be reduced to less than 10%.

Papaverine identified as an inhibitor of high mobility group box 1/receptor for advanced glycation end-products interaction suppresses high mobility group box 1-mediated inflammatory responses.

The interaction of high mobility group box 1 (HMGB1), which is secreted from immune and dying cells during cellular infection and injury, and receptor for advanced glycation end-products (RAGE) appears to be critical for acute and chronic inflammatory disorders. Here we designed a unique cyclic ß-hairpin peptide (Pepb2), which mimics the predicted RAGE-binding domain of HMGB1. Pepb2 competitively inhibited HMGB1/RAGE interaction. We then identified papaverine as a Pepb2 mimetic by in silico 3D-structural similarity screening from the DrugBank library. Papaverine was found to directly inhibit HMGB1/RAGE interaction. It also suppressed the HMGB1-mediated production of pro-inflammatory cytokines, IL-6 and TNF-α, in mouse macrophage-like RAW264.7 cells and bone marrow-derived macrophages. In addition, papaverine attenuated mortality in cecal ligation puncture-induced sepsis model mice. Taken together, these findings indicate that papaverine could become a useful therapeutic against HMGB1/RAGE-mediated sepsis and other inflammatory diseases.

Calculations of Electron Inelastic Mean Free Paths. XI. Data for Liquid Water for Energies from 50 eV to 30 keV.

We calculated electron inelastic mean free paths (IMFPs) for liquid water from its optical energy-loss function (ELF) for electron energies from 50 eV to 30 keV. These calculations were made with the relativistic full Penn algorithm (FPA) that has been used for previous IMFP and electron stopping-power calculations for many elemental solids. We also calculated IMFPs of water with three additional algorithms: the relativistic single-pole approximation (SPA), the relativistic simplified SPA, and the relativistic extended Mermin method. These calculations were made using the same optical ELF in order to assess any differences of the IMFPs arising from choice of the algorithm. We found good agreement among the IMFPs from the four algorithms for energies over 300 eV. For energies less than 100 eV, however, large differences became apparent. IMFPs from the relativistic TPP-2M equation for predicting IMFPs were in good agreement with IMFPs from the four algorithms for energies between 300 eV and 30 keV but there was poorer agreement for lower energies. We calculated values of the static structure factor as a function of momentum transfer from the FPA. The resulting values were in good agreement with results from first-principles calculations and with inelastic X-ray scattering spectroscopy experiments. We made comparisons of our IMFPs with earlier calculations from authors who had used different algorithms and different ELF data sets. IMFP differences could then be analyzed in terms of the algorithms and the data sets. Finally, we compared our IMFPs with measurements of IMFPs and of a related quantity, the effective attenuation length (EAL). There were large variations in the measured IMFPs and EALs (as well as their dependence on electron energy). Further measurements are therefore required to establish consistent data sets and for more detailed comparisons with calculated IMFPs.

Extended Mermin method for calculating the electron inelastic mean free path.

We propose an improved method for calculating electron inelastic mean free paths (IMFPs) in solids from experimental energy-loss functions based on the Mermin dielectric function. The "extended Mermin" method employs a nonlimited number of Mermin oscillators and allows negative oscillators to take into account not only electronic transitions, as is common in the traditional approaches, but also infrared transitions and inner shell electron excitations. The use of only Mermin oscillators naturally preserves two important sum rules when extending to infinite momentum transfer. Excellent agreement is found between calculated IMFPs for Cu and experimental measurements from elastic peak electron spectroscopy. Notably improved fits to the IMFPs derived from analyses of x-ray absorption fine structure measurements for Cu and Mo illustrate the importance of the contribution of infrared transitions in IMFP calculations at low energies.

Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse ES cells to DNA damaging agents.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg(+/-) and Parg(-/-)) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg(-/-) embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg(+/+) ES cells. Parg(-/-) ES cells exhibited the same growth rate as Parg(+/+) ES cells in culture. Sensitivity of Parg(-/-) ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (Parg(+/+)) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.

Stage-specific expression of DNasegamma during B-cell development and its role in B-cell receptor-mediated apoptosis in WEHI-231 cells.

Here, we describe the non-redundant roles of caspase-activated DNase (CAD) and DNasegamma during apoptosis in the immature B-cell line WEHI-231. These cells induce DNA-ladder formation and nuclear fragmentation by activating CAD during cytotoxic drug-induced apoptosis. Moreover, these apoptotic manifestations are accompanied by inhibitor of CAD (ICAD) cleavage and are abrogated by the constitutive expression of a caspase-resistant ICAD mutant. No such nuclear changes occur during oxidative stress-induced necrosis, indicating that neither CAD nor DNasegamma functions under necrotic conditions. Interestingly, the DNA-ladder formation and nuclear fragmentation induced by B-cell receptor ligation occur in the absence of ICAD cleavage and are not significantly affected by the ICAD mutant. Both types of nuclear changes are preceded by the upregulation of DNasegamma expression and are strongly suppressed by 4-(4,6-dichloro-[1, 3, 5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), which is a specific inhibitor of DNasegamma. Our results suggest that DNasegamma provides an alternative mechanism for inducing nuclear changes when the working apoptotic cascade is unsuitable for CAD activation.

cDNA cloning and functional characterization of Xenopus laevis DNase gamma.

We report here the cDNA cloning and functional analysis of Xenopus DNase gamma (xDNase gamma). Two forms of cDNAs are isolated from adult spleen: one composing a 933 bp open reading frame for the enzymatically active xDNase gamma protein, and the other encoding an inactive short alternative form. Northern blot analysis revealed that the xDNase gamma mRNA is expressed in spleen, liver, testis, and ovary. xDNase gamma expression is scarcely detected in the tail muscle of tadpoles; however, it increases during metamorphosis and reaches a maximum during the late metamorphic climax. The ectopic expression of xDNase gamma results in the appearance of extensive DNA fragmentation in C2C12 myoblasts after the induction of apoptosis. In contrast, Xenopus DNase I fails to induce apoptotic DNA ladder formation under the same conditions. Our results suggest a possible involvement of xDNase gamma in apoptosis during amphibian metamorphosis.

Differential apoptotic pathways in human keratinocyte HaCaT cells exposed to UVB and UVC.

The induction of apoptosis in keratinocytes by ultraviolet (UV)-irradiation is considered to be a protective function against skin cancer. UV-induced DNA damage is a crucial event in UVB- and UVC-mediated apoptosis. However, the differences between the UVB- and UVC-induced apoptotic pathways remain unclear. Here we examine the differential mechanisms by which UVB and UVC irradiations induce keratinocyte apoptosis using human keratinocyte HaCaT cells. Differences in the production of (6-4)photoproducts ((6-4)PPs) and cyclobutane pyrimidine dimers (CPDs) were measured following irradiation with UVB and UVC at doses causing the same extent of apoptotic cell death. In addition, main apoptotic features, such as caspase activation and its regulation, were compared between UVB- and UVC-induced apoptosis. Exposures of 500 J/m(2) UVB and 100 J/m(2) UVC resulted in apoptosis to almost the same extent. At these apoptotic doses, the amounts of both (6-4)PPs and CPDs were significantly larger in the case of UVC irradiation than UVB irradiation; in parallel, the release of cytochrome c and Smac/DIABLO and the activation of caspases-9 following UVC irradiation were greater than after UVB irradiation. Importantly, caspase-8 activation occurred only in UVB-irradiated cells. Furthermore, the activation of caspase-8 was not inhibited by caspases-9 and -3 specific tetrapeptide inhibitors, indicating that the caspase-8 cleavage is not due to feedback from activation of caspases-9 and -3. Thus, these results clearly suggest that the reason apoptosis is induced to the same extent by UVB irradiation as by UVC irradiation, despite the lower production of photoproducts in DNA by UVB irradiation, is attributable to the additional activation of the caspase-8 pathway. Thus, UVB irradiation induces apoptosis through both mitochondrial (intrinsic) and caspase-8 activation (extrinsic) pathways, while UVC induces apoptosis only via the intrinsic pathway.

Identification of a novel gene family, paralogs of inhibitor of apoptosis proteins present in plants, fungi, and animals.

Only few orthologs of animal apoptosis regulators have been found in plants. Recently, the ectopic expression of mammalian inhibitor of apoptosis proteins (IAPs) has been shown to affect plant programmed cell death. Here, we identified two novel proteins homologous to Arabidopsis thaliana IAP-like protein (AtILP) 1 and 2 by applying an improved motif searching method. Furthermore, homologs of AtILP1 were found to occur as a novel gene family in other organisms such as fungi and animals including Homo sapiens (HsILP1). Like baculovirus IAP repeats (BIRs) in IAPs, ILPs contain two highly conserved BIR-like domains (BLDs) with a putative C2HC-type zinc finger. Phylogenetic analyses indicated that ILPs are putative paralogs of IAPs. Homology modeling revealed that the three-dimensional structure of BLD in HsILP1 is similar to that of BIR. Transient expression of HsILP1 resulted in inhibition of etoposide-induced apoptosis in HEK293 and HeLaS3 cells. These findings suggest that ILPs are conserved in a wide range of eukaryotes including plants, and that their functions are closely related to those of IAPs.

Structure-based design of an agonistic peptide targeting Fas.

A small agonistic peptide FRAP-4 (WEWT, Fas reactive peptide-4) that binds to the human Fas molecule was discovered using our computer screening strategy named the Amino acid Complement Wave (ACW) method, which is based on the complementarities of interacting amino acids between comprehensive testing peptides and a target protein surface pocket. In silico docking studies demonstrated the specific interaction of FRAP-4 with the main Fas ligand (FasL) binding domain in the Fas molecule. An octamer of this peptide produced by carboxyl terminal linkages of polylysine branches (MAP), (FRAP-4)8-MAP, effectively induced apoptosis in human ovarian cancer cell line NOS4 cells that was associated with the activation of caspases-8, -9 and -3, and the cleavage of PARP. Alanine substitution of the N-terminal W in FRAP-4 resulted in complete loss of FasL-mimetic action of (FRAP-4)8-MAP, suggesting that the aromatic functionality at the N-terminal position W appears to play an essentially important role in Fas binding ability. These observations indicate that the FasL-mimetic peptide should serve as an excellent starting point for the design of effective compounds with FasL-mimetic activity. Furthermore, the ACW method for the structure-based design of optimized small peptides against receptor molecules such as Fas could open new avenues for the development of peptide mimetic and nonpeptidic organic forms to generate novel effective pharmaceuticals.

Analysis of the susceptibility of CD57 T cells to CD3-mediated apoptosis.

After stimulation with anti-CD3 antibody in vitro, CD57(+) T cells showed a greater susceptibility to apoptosis than CD57(-)alphabetaT cell receptor (TCR)(+) T cells (regular alphabeta T cells). The apoptotic fraction of CD57(+) T cells showed an increased production of active caspase-3. An increase in both Fas expression and Fas-ligand (FasL) production was also observed in CD57(+) T cells, whereas the expression of survivin was suppressed in CD57(+) T cells compared to that of regular alphabeta T cells. CD57(+) T cells display a biased expansion of a few Vbeta T cell fractions in individuals, but such Vbeta T cells were not specifically susceptible to CD3-mediated apoptosis. The TCR expression level of CD57(+) T cells was much lower than that of regular T cells and anti-TCR antibody stimulation induced a smaller apoptotic proportion of CD57(+) T cells than did anti-CD3 antibody. Although the CD3epsilon expression levels were similar in both T cell subsets, the CD3zeta level of CD57(+) T cells was significantly higher than that of regular T cells. These results suggest that several apoptotic and anti-apoptotic molecules are involved in the CD3-induced apoptosis of CD57(+) T cells and raise the possibility that the imbalance in expression of the CD3epsilon and CD3zeta chains may also contribute to the susceptibility of CD57(+) T cells to undergo apoptosis.

Differential DNases are selectively used in neuronal apoptosis depending on the differentiation state.

In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase gamma, in neuronal apoptosis. High expression of CAD, but not DNase gamma, is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspase-resistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase gamma occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase gamma caused by its antisense RNA. The induction of DNase gamma is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase gamma. These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase gamma depending on the differentiation state. Furthermore, DNase gamma is suggested to be involved in naturally occurring apoptosis in developing nervous systems.

Sustained release of Smac/DIABLO from mitochondria commits to undergo UVB-induced apoptosis.

Apoptotic response of keratinocytes to UVB irradiation has physiological significance on photocarcinogenesis. Here, we show that the sustained release of Smac/DIABLO from mitochondria is an important event for the onset of apoptosis in keratinocytes exposed to UVB irradiation. In human keratinocyte HaCaT cells, UVB irradiation at 500 J/m(2), but not at 150 J/m(2), induces apoptosis. Significant activations of caspases-9 and -3, and slight activation of caspase-7 were observed only in 500 J/m(2) UVB irradiated HaCaT cells. Correspondingly, the cleavage of PARP, a substrate of caspases-3 and -7, was detected in cells irradiated at 500 J/m(2) UVB, but not at 150 J/m(2). However, with both 150 and 500 J/m(2) UVB irradiation, cytochrome c, an activator of caspase-9 via the formation of apoptosome, was released from mitochondria to the cytosol at the same extent. In contrast, significant amounts of Smac/DIABLO are released from mitochondria to the cytosol only with 500 J/m(2) UVB irradiation, and that the level of XIAP is decreased. These results suggest that the extent of Smac/DIABLO efflux from mitochondria is a determinant whether a cell will undergo apoptosis or survival.

Activation of caspase-8 is critical for sensitivity to cytotoxic anti-Fas antibody-induced apoptosis in human ovarian cancer cells.

Two ovarian cancer cell lines named NOS4 and SKOV-3 have been shown to have different sensitivities to a cytotoxic anti-Fas antibody, CH-11. Although both cell lines express Fas molecules on the cell surfaces at the same intensities, apoptosis is induced by CH-11 in NOS4 cells but not in SKOV-3 cells. In this study, the different apoptosis-sensitivities of these cells were assessed. Both cell lines express almost the same levels of FADD, RIP, c-FLIP, FAP-1, Bax, Bcl-2 and Bcl-XL. Evidence of caspase-8, caspase-9 and caspase-3 activation and of cleavage of PARP and Bid was obtained in NOS4 cells but not in SKOV-3 cells. When triggered by FasL protein, DNA fragmentation and caspase-8 activation were observed in SKOV-3 cells, though they were not as clear as in NOS4 cells. All the anti-Fas antibody-mediated signals for apoptosis induction in NOS4 cells were completely blocked by a caspase-8-specific inhibitor, Z-IETD-FMK. These results indicate that the different sensitivities to the anti-Fas antibody are solely dependent on the activation of caspase-8, which could be influenced by yet unknown qualitative or quantitative abnormalities in molecules involved in DISC formation.

Isolation and characterization of the DLAD/Dlad genes, which lie head-to-head with the genes for urate oxidase.

We previously found that a novel DNA endonuclease named DLAD (DNase II-Like Acid DNase) is specifically expressed in murine liver. Here, we describe the isolation and characterization of the human DLAD and mouse Dlad genes. Both DLAD and Dlad consist of 6 exons. DLAD encodes a 361 amino acid protein sharing 34.6% amino acid identity with human DNase II. Although a recombinant protein for the putative human DLAD has a divalent cation-independent acid DNase activity, expression of the DLAD mRNA containing the entire open reading frame was not detected in any human tissues tested, except for lung, in which a short 1.1 kb transcript lacking the first two exons is expressed. Interestingly, sequence analysis of Dlad showed that the 1st exon of the urate oxidase gene, Uox, is located on the opposite strand in its 5'-flanking region. The head-to-head organization of DLAD and UOX is conserved in the human genomic sequence. Promoter analysis revealed that the intergenic region between Dlad and Uox has promoter activity for both the Dlad and Uox directions, however, the corresponding human genomic fragment has promoter activity only for DLAD. It is known that murine Uox is expressed only in the liver, whereas human UOX has been inactivated as a pseudogene. On the basis of these results, the expression of DLAD/Dlad and UOX/Uox is suggested to be coordinated by a common regulatory mechanism(s), and the balance between the two enzymes is thought to be important for maintaining the purine nucleotide pool in the liver.

[Swallowing rehabilitation in two elderly patients with cerebral infarction].

We report two cases of cerebral infarction in which swallowing function improved following swallowing rehabilitation. Patient 1 was an 82-year-old man, who was admitted due to rheumatoid arthritis and multiple cerebral infarction, suffering from aspiration pneumonia. The abnormality of swallowing was assessed by the water swallowing test and videofluorography. It has been reported that videofluorography is useful in the diagnosis of aspiration. Three weeks after the start of swallowing rehabilitation, the serum level of inflammatory markers and the chest X-ray had returned to normal. His score on the water swallowing test had improved. Patient 2 was a 68-year-old [correction of 62] man, who was admitted with severe hemiplegia, dysphagia and dysarthria. One month after the swallowing rehabilitation, videofluorography showed that the magnitude of aspiration into the trachea had decreased and the pooling of barium in the piriform sinus had disappeared. The patient could begin taking a little food by mouth. These results suggest that swallowing rehabilitation will be affect the clinical improvement of swallowing function and help preventing aspiration pneumonia in our hospital.

DNA fragmentation in ischemic core and penumbra in focal cerebral ischemia in rats.

Although apoptotic cell death has been suggested to be involved in ischemic injury of the brain, the precise mechanisms of ischemic neuronal cell death are unknown. Here, we examined the biochemical feature of apoptosis (i.e. DNA fragmentation) in male spontaneously hypertensive rats (5-7 months old) subjected to photothrombotic distal middle cerebral artery (MCA) occlusion. After MCA occlusion, the brain was cut in a cryostat to produce a standard coronal block and samples were dissected from the regions corresponding to the ischemic core, penumbra and contralateral control areas. Changes in cerebral blood flow (CBF) were monitored at 1 mm posterior and 2-4 mm lateral to the bregma by means of a laser-Doppler flowmetry. After MCA occlusion, CBF was decreased to 72+/-18 (+/-S.D.), 50+/-14, and 35+/-11% of the control values at 2, 3, and 4 mm from the midline, respectively. DNA fragmentation characteristics of apoptosis were examined in these samples by conventional and pulse-field gel electrophoresis. On the conventional gel electrophoresis, nucleosomal DNA fragmentation was detected in the penumbral zone at 6 h after MCA occlusion. Large DNA fragments of 50 and 20 kbp were detected in the penumbral zone and also in the ischemic core region at 3 h after distal MCA occlusion. The large DNA fragments seen on the pulse-field gel elecrophoresis were further degraded to small DNA fragments at 6 h after MCA occlusion in the penumbral zone but not in the core regions. The evolving DNA fragmentation was observed between 3 and 6 h after the onset of brain ischemia in the penumbra, suggesting that apoptosis may contribute to the development of ischemic infarction.

Seaweed prevents breast cancer?

To investigate the chemopreventive effects of seaweed on breast cancer, we have been studying the relationship between iodine and breast cancer. We found earlier that the seaweed, wakame, showed a suppressive effect on the proliferation of DMBA (dimethylbenz(a)anthracene)-induced rat mammary tumors, possibly via apoptosis induction. In the present study, powdered mekabu was placed in distilled water, and left to stand for 24 h at 4 degrees C. The filtered supernatant was used as mekabu solution. It showed an extremely strong suppressive effect on rat mammary carcinogenesis when used in daily drinking water, without toxicity. In vitro, mekabu solution strongly induced apoptosis in 3 kinds of human breast cancer cells. These effects were stronger than those of a chemotherapeutic agent widely used to treat human breast cancer. Furthermore, no apoptosis induction was observed in normal human mammary cells. In Japan, mekabu is widely consumed as a safe, inexpensive food. Our results suggest that mekabu has potential for chemoprevention of human breast cancer.

Characterization of human DNase I family endonucleases and activation of DNase gamma during apoptosis.

We describe here the characterization of the so far identified human DNase I family DNases, DNase I, DNase X, DNase gamma, and DNAS1L2. The DNase I family genes are found to be expressed with different tissue specificities and suggested to play unique physiological roles. All the recombinant DNases are shown to be Ca(2+)/Mg(2+)-dependent endonucleases and catalyze DNA hydrolysis to produce 3'-OH/5'-P ends. High activities for DNase I, DNase X, and DNase gamma are observed under neutral conditions, whereas DNAS1L2 shows its maximum activity at acidic pH. These enzymes have also some other peculiarities: different sensitivities to G-actin, aurintricarboxylic acid, and metal ions are observed. Using a transient expression system in HeLa S3 cells, the possible involvement of the DNases in apoptosis was examined. The ectopic expression of each DNase has no toxic effect on the host cells; however, extensive DNA fragmentation is observed only in DNase gamma-transfected cells after the induction of apoptosis. Furthermore, DNase gamma is revealed to be located at the perinuclear region in living cells, and to translocate into the nucleus during apoptosis. Our results demonstrate that DNase I, DNase X, DNase gamma, and DNAS1L2 have similar but unique endonuclease activities, and that among DNase I family DNases, DNase gamma is capable of producing apoptotic DNA fragmentation in mammalian cells.

[The effects of pulmonary rehabilitation with chronic obstructive pulmonary disease (COPD)].

It is controversial whether pulmonary rehabilitation is effective in patients with chronic obstructive pulmonary disease (COPD). To test the effect of pulmonary rehabilitation, 7 patients with COPD (aged 76.0 +/- 2.6 years) were enrolled in pulmonary rehabilitation program for 6 weeks. The program consisted of relaxation, pursed lip breathing, diaphragmatic breathing, panic control, muscle stretch gymnastics, and exercise training. The distance of the 6-minute walking test increased significantly from 246.4 +/- 38.0 (m) to 304.3 +/- 28.4 (m) (p < 0.05). The minimum SpO2 during the 6-minute walking test increased from 86.0 +/- 2.8 (%) to 90.1 +/- 1.3 (%) and dyspnea as measured with Borg scale decreased from 5.6 +/- 1.1 to 4.6 +/- 0.5, although they were not significantly different. These results suggest that pulmonary rehabilitation might improve exercise tolerance in elderly patients with COPD.