CCL17 and CCL22 chemokines within tumor microenvironment are related to infiltration of regulatory T cells in esophageal squamous cell carcinoma.

It has been reported that an increased population of regulatory T cells (T-regs) is one of the reasons for impaired anti-tumor immunity. We investigated the frequency of Foxp3(+) T-regs in tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) of patients with esophageal squamous cell carcinoma (ESCC). Furthermore, in order to elucidate the mechanisms behind T-regs accumulation within tumors, we evaluated the relationship between CCL17 or CCL22 expression and the frequency of Foxp3(+) T-regs. CD4(+)CD25(+)Foxp3(+) T-regs as a percentage of CD4(+) cells were counted by flow cytometry. The frequency of CCL17(+) or CCL22(+) cells among CD14(+) cells in tumors was also evaluated by flow cytometry. Moreover, an in vitro migration assay using T-regs derived from ESCC was performed in the presence of CCL17 or CCL22. The frequency of Foxp3(+) T-regs in TILs was significantly higher than that in the normal esophageal mucosa (24.6 +/- 10.0 vs 7.1 +/- 5.9%, P < 0.01). The frequency of Foxp3(+) T-regs in PBLs of ESCC patients was significantly higher than that in normal healthy donors (7.0 +/- 4.2 vs 2.5 +/- 1.0%, P < 0.01). Furthermore, the frequency of CCL17(+) or CCL22(+) cells among CD14(+) cells within tumors was significantly higher than that of normal esophageal mucosa, and there was a significant correlation between the frequency of CCL17(+) or CCL22(+) cells and Foxp3(+) T-regs in TILs. In addition, the in vitro migration assay indicated that T-regs were significantly induced to migrate by CCL17 or CCL22. In conclusion, CCL17 and CCL22 within the tumor are related to the increased population of Foxp3(+) T-regs in ESCC.

NK cell dysfunction with down-regulated CD16 and up-regulated CD56 molecules in patients with esophageal squamous cell carcinoma.

NK cells can be divided into two subsets, CD56(dim) and CD56(bright) NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed NK cell dysfunction in patients with esophageal squamous cell carcinoma (ESCC), with a particular focus on the expression of CD16 and CD56 molecules. Expression of CD16 and CD56, and the distribution of CD56(dim) or CD56(bright) NK cells gated on CD56(+)CD3(-) NK cells were compared between ESCC patients (n= 40) and healthy donors (n= 38). Purified NK cells were evaluated for Cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against epidermal growth factor receptor (EGFR)-expressing ESCC cell lines. Although there were no significant differences in the distribution of CD56(dim) and CD56(bright) NK cells between ESCC patients and healthy donors, down-regulated CD16 and up-regulated CD56 were significantly observed on NK cells of ESCC patients, paralleling the impairment of Cetuximab-mediated ADCC, in comparison with healthy donors. After patients received curative resections of ESCC, the down-regulated CD16 and up-regulated CD56 were significantly restored to the levels of healthy donors. Moreover, TGF-beta1 partially contributed to down-regulation of CD16 on NK cells. Down-regulated CD16 and up-regulated CD56 molecules on NK cells were observed in ESCC patients, resulting in NK cell dysfunction.

Exercise in, and adaptations to a cold environment have no effect on SIgA.

AIM: The authors hypothesized that inconsistent SIgA response to exercise is caused by the different adaptative status of subjects to a cold environment. The purposes of the study were to examine whether moderate-intense exercise in a cold environment decreases SIgA and whether adaptation to a cold environment has any effect on SIgA. METHODS: Young male skaters, short track (N=9) and inline (N=10), participated in this study. All subjects cycled for 60 min at 65% VO(2max) in cold (ambient temperature: 5 +or - 1 degrees Celsius, relative humidity 41 + or - 9%) and thermoneutral (ambient temperature: 21 + or - 1 degrees Celsius, relative humidity 35 + or - 5%) conditions. Saliva samples were collected as follows: before and after 1hour of environmental exposure; immediately, 30-min, 60-min and 120-min after the exercise. RESULTS AND CONCLUSIONS: Salivary SIgA and saliva flow rate decreased after the exercise in both groups only in thermoneutral conditions. The SIgA secretion rate did not decrease after moderate-high intensity exercise in a cold environment, and the SIgA response to exercise was not affected by the different adaptative status of subjects to the cold environment.

Regulation of the yeast Yap1p nuclear export signal is mediated by redox signal-induced reversible disulfide bond formation.

Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H(2)O(2) or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H(2)O(2) and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H(2)O(2) or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H(2)O(2) and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H(2)O(2)-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys(598)) and second (Cys(620)) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

Concentration-dependent effects of salicylaldoxime on chloroplast reactions.

Salicylaldoxime has been found to have a variety of concentration-dependent effects on chloroplast activities. At low concentrations (less than 10 mM), salicyladoxime reversibly inhibits all reactions which involve Photosystem II. Since the DCMU-insensitive silicomolybdate Hill reaction is also inhibited, one site of inhibition is definitely located before the DCMU-sensitive site, possibly before the photoact. The inhibition kinetics and the response of chloroplast fluorescence may indicate another site in the DCMU-sensitive region. At almost exactly the same concentrations (less than 10 mM), salicylaldoxime uncouples phosphorylation reversibly, whether it is supported by Photosystem II or by Photosystem I. At higher concentrations (approx. 20 mM) salicylaldoxime inhibits Photosystem II irreversibly, uncouples irreversibly, and begins to cause changes in chloroplast light scattering which could be manifestations of membrane damage. At very high concentrations (approx. 45 mM) salicylaldoxime irreversibly inhibits Photosystem I activity in the region of plastocyanin. This is indicated by the ability of salicylaldoxime to inhibit the photooxidation of cytochrome f but not the photooxidation of P-700.

Pathways of silicomolybdate photoreduction and associated photophosphorylation in tobacco chloroplasts.

Three sites of silicomolybdate reduction in the electron transport chain of isolated tobacco chloroplasts are described. The relative participation of these sites is greatly influenced by the particular reaction conditions. One site (the only site when the reaction medium contains high concentrations of bovine serum albumin (greater than 5 mg/ml) is associated with Photosystem I, since it supports phosphorylation with a P/e2 value close to 1 and the reaction is totally sensitive to both plastocyanin inhibitors and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Two other sites of silicomolybdate reduction are associated with Photosystem II. One site is 3-(3,4-dichlorophenyl)-1,1-dimethylurea insensitive and supports phosphorylation when the reaction mixture contains dimethyl sulfoxide and glycerol (protective agents). The P/e2 value routinely observed is about 0.2. Bovine serum albumin (1-2 mg/ml) can also act as a protective agent, but the efficiency of Photosystem II phosphorylation observed is lower. Silicomolybdate reduction supports virtually no phosphorylation, regardless of the reduction pathway, when the reaction mixture contains no protective agents. This is due to irreversible uncoupling by silicomolybdate itself. The silicomolybdate uncoupling is potentiated by high salt concentrations even if the presence of protective agents. Exposure of chloroplasts to silicomolybdate in the absence of protective agents rapidly inactivates both photosystems.

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).

The role of chloride ion in photosystem II. I. Effects of chloride ion on photosystem II electron transport and on hydroxylamine inhibition.

1. Chloroplasts washed with Cl--free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl- is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems. 2. Strong Cl--dependence of Hill activity was observed invariably at all pH values tested (5.5--8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll). 3. In the absence of added Cl- the functionally Cl--depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H2O2 at rates which are 4--12 times faster than the rate of the residual Hill reaction. 4. The Cl--concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl- in equilibrium E . Cl- (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation. 5. The initial phase of the development of inhibition of water oxidation in Cl--depleted chloroplasts during the dark incubation with NH2OH (1/2 H2SO4) is 5 times slower when the incubation medium contains Cl- than when the medium contains NH2OH alone or NH2OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O2 evolution).

Molecular identification of glutathione synthetase (GSH2) gene from Saccharomyces cerevisiae.

The hypothetical protein YOL049w on the chromosome XV was identified to be the structural gene for glutathione synthetase (GSH2) of Saccharomyces cerevisiae. Translational initiation site was identified by making the GSH2-lacZ fusion. The GSH2 gene contained an open reading frame (1473 bp) with 491 amino acids, and molecular weight of the GSH2 gene product was calculated to be 55,812. Glutathione synthetase activity in transformant carrying the GSH2 gene with multicopy plasmid increased approximately 4-fold. The GSH2 gene was not essential for growth of yeast cell, and glutathione was not detected from the gsh2 disrupter.

F0F1-ATPase of Vibrio parahaemolyticus: purification using new detergents and characterization.

Previous attempts to isolate a stable F0F1-ATPase complex (H(+)-translocating ATPase) from Vibrio parahaemolyticus have been unsuccessful. Using new non-ionic detergents (alkyl thiomaltosides), a stable F0F1 complex with a high specific activity (15-25 units/mg protein) was purified and characterized. The purified F0F1-ATPase consists of eight subunits (alpha, beta, gamma, delta, epsilon, a, b and c). The new detergents, in combination with sucrose (or glycerol), lipid, dithiothreitol and phenylmethylsulfonyl fluoride, effectively stabilized the F0F1 complex. The ATPase activity of the F0F1 complex was greatly increased by anions, such as SO4(2-) and SO3(2-). Sodium ion increased the activity by about 2-fold. Dicyclohexylcarbodiimide, Zn2+, 4-acetamido-4'-isothiocyanostilben-2,2'disulfonate and tetrachlorosalicylanilide inhibited F0F1-ATPase activity. Ethanol, which stimulated F1-ATPase activity, inhibited F0F1-ATPase activity. Methanol, Na3VO4 and bafilomycin A1 did not have any significant effect on F0F1-ATPase activity, although methanol, like ethanol, stimulated F1-ATPase activity.

Oxidative stress response in yeast: glutathione peroxidase of Hansenula mrakii is bound to the membrane of both mitochondria and cytoplasm.

The yeast Hansenula mrakii IFO 0895 induces glutathione peroxidase (GPx) when the cells are exposed to the oxidative stress such as lipid hydroperoxide, superoxide- and hydroxy radical-generating conditions. To clarify the localization of GPx in H. mrakii cell, distribution of the enzyme was investigated. After centrifugation of the yeast protoplast homogenates at 2500 x g for 10 min, 67% of total GPx activity was recovered from the supernatant (Sup. 1) and 33% was from the pellet (Pellet 1). When the Sup. 1 was fractionated by sucrose density gradient ultracentrifugation, GPx activity was essentially recovered from the mitochondria fraction. Submitochondrial localization of the enzyme showed that 95% and 2.5% of the enzyme was recovered from the inner and outer membrane, respectively. No GPx activity was detected neither in intermembrane space nor in matrix of mitochondria. On the other hand, at least 12% of total GPx activity was recovered from the purified plasma membrane which was obtained from the Pellet 1 by successive sucrose density gradient centrifugation. Thus, the GPx of H. mrakii is present in the inner and outer membrane of mitochondria as well as the plasma membrane.

A novel immunomodulator KRP-203 combined with cyclosporine prolonged graft survival and abrogated transplant vasculopathy in rat heart allografts.

To find more effective and less toxic immunosuppressive strategies in long-term treatment for organ transplantation patients, we examined the effects on rat heart allograft survival of a novel sphigosine-1-phosphate receptor agonist, KRP-203, combined with a subtherapeutic dose of cyclosporine (CsA). Rat heart transplantation was performed across a major histocompatibility complex-incompatible (DA to LEW) rat combination. KRP-203 alone showed little or no effect on heart allograft survival. In contrast, KRP-203 combined with a subtherapeutic dose of CsA led to prolonged allograft survival. Histologic analyses showed that the combination completely suppressed acute rejection, as characterized by allograft vasculopathy, mononuclear cell infiltration, and myocardial necrosis in the heart allografts. RT-PCR analysis showed that the allografts treated with CsA or KRP-203 alone showed no suppression of IL-10, IFN-gamma, and TNF-alpha mRNA expression, but when combined with a subtherapeutic dose of CsA it completely suppressed their mRNA expressions. Furthermore, the combination treatment reduced donor-specific antibody production. KRP-203 combined with a subtherapeutic dose of CsA synergistically prolonged rat heart allograft survival. The combination of CsA with KRP-203 may provide an option to prevent allograft rejection and reduce adverse effects.

Interferon-beta induction/interferon-alpha2b plus ribavirin therapy in patients with chronic hepatitis C.

Treatment of chronic hepatitis C virus (HCV) infection with interferon (IFN) and ribavirin improves the rate of eradication of the virus by less than 20% in patients with genotype 1b and a high viral load. In this study we assessed whether IFN-beta induction/IFN-alpha2b plus ribavirin enhances the efficacy of the therapy in patients with chronic hepatitis C. The efficacy of IFN-beta induction/IFN-alpha2b plus ribavirin therapy (group A, n=7) was compared with that of IFN-alpha2b plus ribavirin (group B, n=7) in 14 patients with high levels of HCV-RNA (> 100 K/U/ml). No significant differences were observed in the clearance of HCV-RNA between the two groups (A and B, respectively) 2 weeks after the start of the treatment (0% and 14.3%), at the end of the treatment (71.4% and 100%) and 6 months after the end of the treatment (28.6% and 14.3%). Recovery was complete in 28.6% and 14.3%, transient in 42.9% and 85.7% and absent in 28.6% and 0% in groups A and B, respectively. Early log changes in the viral load from the baseline after 2 weeks of treatment were 2.41 +/- 0.91 and 2.77 +/- 0.20 in groups A and B, respectively, with no significant difference between the two groups. In the present study, we were not able to demonstrate that IFN-beta induction/IFN-alpha2b plus ribavirin therapy was superior to IFN-alpha2b plus ribavirin therapy in patients with genotype 1b and high viral loads.

Oxidative stress response in yeast: effect of glutathione on adaptation to hydrogen peroxide stress in Saccharomyces cerevisiae.

Role of intracellular glutathione in the response of Saccharomyces cerevisiae to H2O2 was investigated. Depletion of cellular glutathione or inhibition of gamma-glutamylcysteine synthetase (GSH-I) enhanced the sensitivity to H2O2 and suppressed the adaptation to H2O2. A mutant deficient in GSH-I also showed the hypersensitivity and could not adapt to H2O2. Incubation of the cell with amino acids constituting glutathione (L-Glu, L-Cys, Gly) increased the intracellular glutathione content, and subsequently the cell acquired resistance against H2O2. These results strongly suggest that intracellular glutathione plays an important role in the adaptive response in S. cerevisiae to oxidative damage.

Effects of alcohols on the hydrolysis of colominic acid catalyzed by Streptococcus neuraminidase.

Regulation of the activity of neuraminidase of Streptococcus sp. (group K) was evaluated by examining the effects of alcohols on the hydrolysis of colominic acids catalyzed by the neuraminidase. Two kinds of alcohol binding site, activation and inhibition sites, were proposed to exist. Competitive inhibition was observed with alcohols smaller than polyethylene glycol #300 (average molecular weight: 300), so the inhibition site is considered to be the substrate binding site, the size of which was estimated to be 10 A in diameter. On the contrary, polyethylene glycols larger than this size activated the enzyme by 1.5-1.8 times. The activity could be raised by binding of the polyethylene glycols to the activation site. This activation was shown to be due solely to the decrease in the Michaelis constant, Km. The smaller polyethylene glycols (#200 and #300) were also considered to bind to the activation site, although activation was not clearly observed due to compensation with inhibition. Strong substrate inhibition by colominic acid was also observed. The activity of Streptococcus neuraminidase was shown to be regulated intricately by the substrate colominic acid and alcohols contained in the reaction medium.

Introduction of a series of alkyl thiomaltosides, useful new non-ionic detergents, to membrane biochemistry.

We synthesized a series of non-ionic detergents, alkyl thiomaltosides, and investigated their properties and usefulness. We solubilized membrane proteins of Vibrio parahaemolyticus using the detergents. With octyl thiomaltoside, nonyl thiomaltoside, decyl thiomaltoside, or undecyl thiomaltoside, we observed satisfactory solubilization of the membrane proteins. Alkyl thiomaltosides possessing longer alkyl chains showed better solubilization than ones possessing shorter chains. H(+)-translocating ATPase (F0F1), which is localized in the cytoplasmic membrane (inner membrane), was solubilized with the detergents, and the solubilized enzyme showed much higher specific activity than that solubilized with octyl glucoside or heptyl thioglucoside, other useful non-ionic detergents. 5'-Nucleotidase, which seems to be an outer membrane protein, was also efficiently solubilized with the alkyl thiomaltosides. Membrane proteins of Escherichia coli were also efficiently solubilized with the detergents. Octyl thiomaltoside and nonyl thiomaltoside were removed fairly rapidly on dialysis. Decyl thiomaltoside was removed slowly, and undecyl thiomaltoside and dodecyl thiomaltoside were difficult to remove by dialysis.

Activity of neurons in the nucleus of the solitary tract of rats: effect of osmotic and mechanical stimuli.

Patch-clamp recordings were used to examine the osmosensitivity and mechanosensitivity of neurons in the caudal part of the nucleus tractus solitarius in coronal slices from rat brain. Firing rates and membrane potentials were measured as slices were exposed to perfusate which varied in its osmolality and/or sodium concentration. In all cells tested, the responses to change in the sodium concentration of perfusate were duplicated by osmolality changes of sucrose or mannitol. When nucleus tractus solitarius cells were tested with changes in pressure applied via the pipette, responses to positive or negative pressure paralleled their responses to osmotic stimulation. We suggest that a mechanosensitive receptor exists on osmosensitive neurons within the nucleus tractus solitarius, and this receptor may be responsible for changes in the firing rate and membrane potential which occur in the nucleus tractus solitarius neurons.

Characteristics of the chemical forms of 11C, 13N, and 15O induced in air by the operation of a 100 MeV electron linear accelerator.

To characterize airborne radioactivity induced by the operation of high-energy accelerators, the fractions of aerosol and gaseous components, and the chemical forms of 11C, 13N, and 15O produced in the air of a target room of a 100 MeV electron linear accelerator were studied. Measurements of radioactivity using a particulate air sampling filter and a gas flow-through ionization chamber showed that more than 98% of 11C, 13N, and 15O were present as gaseous forms. Their chemical forms, detected by means of radio-gas chromatography, were 11C as CO2; 13N as N2 and NO; and 15O as O2 and NO. Machine operating conditions, which affect the compositions of the induced radionuclides and of their chemical forms, and the resulting effect on the estimation of internal doses are discussed.

Factors associated with deterioration of mini nutritional assessment-short form status of nursing home residents during a 2-year period.

OBJECTIVE: A number of other studies have been conducted to verify the Mini Nutritional Assessment (MNA) or the MNA short form (MNA-SF) as a nutritional assessment/screening tool in various clinical settings or communities. However, there are few longitudinal studies using these tools to analyze which factors affect the incidence of deteriorating nutritional status. We tried to identify the factors associated with deterioration of MNA-SF status of nursing home residents during a 2-year period. METHODS: Participants were 392 people with a mean age of 84.3 in 12 nursing homes in Japan. The factors associated with deterioration in MNA-SF categories during the study period compared to stable/improved MNA-SF categories were identified. RESULTS: At baseline, 19.9% of the participants were malnourished and 60.2% were at risk of malnutrition, according to the MNA-SF classification. After 2 years, 66.3% participants maintained and 6.1% participants improved their nutritional status according to the MNA-SF classification, while 27.6% showed deterioration in MNA-SF status. Stepwise logistic-regression procedure indicated that basic ADL impairment and hospitalization during the follow-up period were associated with declining MNA-SF status. CONCLUSIONS: Poor basic ADL status and hospitalization during the follow-up period were associated with malnutrition and risk of malnutrition as assessed by MNA-SF of nursing homes residents during a 2-year period.