14-3-3ζ-Mediated Stimulation of Oxidative Phosphorylation Exacerbates Oxidative Damage Under Hypothermic Oxygenated Conditions in Human Renal Tubular Cells (HK-2).

Cellular survival and death are at least partially regulated by the phosphorylation of proteins. A chaperon protein, 14-3-3ζ, regulates the activity of many proteins by covering the phosphorylation site within a 14-3-3 binding motif. Therefore, regulation of 14-3-3ζ activity may affect the fate of cells subjected to cold preservation and/or hypothermic oxygenated conditions. The present study assessed whether 14-3-3ζ protects cells from hypothermic oxygenation-induced injury and clarified its role in mitochondrial functions. Human renal tubular cell line HK-2 or 14-3-3ζ-overexpressed HK-2 (ζHK-2) cells were subjected to 72 hours of normoxic cold preservation in UW solution with or without antioxidants and hydroperoxides. Cellular death, adenosine triphosphate (ATP) content, and MTT catabolism were evaluated. Deferoxamine treatment reduced cellular death and augmented ATP content in both cell types. These indices were higher in ζHK-2, regardless of deferoxamine treatment. Exposure to hydroperoxides did not affect cellular death in either cell type, whereas hydroperoxide supplementation significantly reduced ATP content, except for low-dose hydrogen peroxide in HK-2 cells. MTT assay at normal state showed higher values in ζHK-2 cells, whereas it was impaired by hydroperoxides in both cell types. These results suggest that accumulation of hydroperoxides as a byproduct of the augmented oxidative phosphorylation by 14-3-3ζ overexpression causes mitochondrial dysfunction. In conclusion, despite possessing many potentially protective functions, 14-3-3ζ exacerbates cellular injury under hypothermic oxygenated conditions. 14-3-3ζ accelerates mitochondrial functions together with iron-dependent oxidative damage. Although further investigations are necessary, upregulation of 14-3-3ζ could be a method to maintain mitochondrial function under hypothermic oxygenated conditions, as shown in hypothermic machine preservation of renal grafts, when appropriate antioxidant treatment is administered.

Protective role of nitric oxide in ischemia and reperfusion injury of the liver.

BACKGROUND: The suppressed production of nitric oxide (NO), associated with endothelial dysfunction, is thought to be a cause of ischemia and reperfusion injury of the liver. But findings of the salutary effects of NO enhancement on such injury have been conflicting. In this study, we tested our hypothesis that NO enhancement would attenuate ischemic liver injury. For this purpose, an NO precursor, L-arginine, and a novel NO donor, FK409, were applied to a 2-hour total hepatic vascular exclusion model in dogs. STUDY DESIGN: L-arginine was administered IV at a dose of 100 mg/kg twice (n = 5), while 300 mg/kg twice of FK409 was infused continuously into the portal vein (n = 5). The drugs were given to the animals for 30 and 60 minutes before and after ischemia, respectively. Non-treated animals were used as the control (n = 10). Two-week survival, systemic and hepatic hemodynamics indices, liver function tests, energy metabolism, and histopathology were analyzed. RESULTS: Both treatments comparably augmented hepatic tissue blood flow, decreased liver enzyme release, and increased high-energy phosphate restoration during the reperfusion period, all of which contributed to rescuing all of the treated animals from the 2-hour total hepatic ischemia. In contrast, ischemia caused 70% mortality in the control group. Histologically, structural abnormality and neutrophil infiltration were markedly attenuated by the treatments. Systemic hypotension was observed in the animals treated with FK409, however. CONCLUSIONS: Our data demonstrate that NO enhancement alleviates the liver injury caused by ischemia and reperfusion. The supplementation of L-arginine, rather than FK409, is considered more applicable to clinical use because of the absence of systemic adverse effects.

[Bactericidal and anti-toxin activities of catechin on enterohemorrhagic Escherichia coli].

We examined the bactericidal activity of catechin, an astringent ingredient of tea, on enterohemorrhagic Escherichia coli (EHEC) O157:H7 and the anti-toxin activity of catechin on vero toxin (VT), the main pathogenic factor of EHEC O157:H7. To examine bactericidal activity, we added 1 X 10(4) CFU/ml bacteria to 1.25 to 20 W/V% of green tea extract or the PBS solution containing 25 to 400 micrograms/ml of (-) epigallocatechin gallate (EGCg), which is the main catechin ingredient of green tea leaf, and counted the number of live bacteria at various intervals. After 3 to 5 hours, no live bacteria were seen in 1.25 to 2.5 (regular drinking concentration) % green tea extract. In the high concentrations of 100 to 400 micrograms/ml EGCg the number of live bacteria decreased with time and after 24 hours no survivors were seen. In the low concentrations of 25 to 50 micrograms/ml EGCg, however, no change was observed in the number of live bacteria during 5 hours. After 24 hours the bacteria in 50 micrograms/ml were killed and the number of bacteria in 25 micrograms/ml decreased to one tenth of that at the start. To examine the anti-toxin activity, we mixed equal volumes of 2 ng/0.1 ml VT2 and 0.5 to 2 mg/0.1 ml catechin in vitro and incubated them at 37 degrees C for various times. Then we inoculated 0.2 ml of the mixture intraperitonealy to BALB/c mice. One mg of catechin inhibited by 100% the lethal toxicity of 2 ng of VT2 (LD 100) to mice. The inhibition of lethal toxicity of VT2 by catechin depended on the incubation time. The rate of inhibition was 0, 40 and 100% for 9, 12 and 18-24 hours incubation, respectively. These results suggest that catechin has not only bactericidal activity on EHEC O157:H7 but also anti-toxin activity on vero toxin.

[SMANCS-TAE combined with PEI in the treatment for hepatocellular carcinoma].

From January 1996 to August 1997, 24 patients with advanced hepatocellular carcinoma (HCC) equal to or more than 2 cm (mean +/- SD; 4.1 +/- 3.0 cm) in main tumor diameter were treated by SMANCS-TAE (20 cases) or SMANCS-TAI (4 cases) combined with PEI. Six cases had solitary lesion, 16 cases had multiple lesions, and 2 cases had massive lesions. After this combination therapy, 21 of 24 cases had complete tumor necrosis. During 3 to 19 months follow up period, 12 cases had cancer-free survival (SMANCS-TAI; 3 cases), and 9 cases had tumor recurrences (3 cases were local recurrences and 6 cases involved new lesions). Two cases died of hepatic infarction and cancer death, however, the remaining 22 cases were surviving. SMANCS-TAE combined with PEI is useful treatment for advanced large or multiple HCC lesions in patients who are poor surgical risks.

[Prophylactic effect of black tea extract as gargle against influenza].

We examined whether gargling with black tea prevents influenza infection. Tests were carried out during a five month period (October 1992 to March 1993). The control group that followed their normal daily routine, whereas the test group that gargled with 0.5 w/v% black tea extract twice daily (at 8 a.m. and 5 p.m.). Influenza viruses were isolated from influenza patients and an antigen analysis was carried out. As a result, two strains of influenza A viruses (H3N2) and ten strains of B virus were detected. An HI test was done using paired sera of the control group and the test group. The HI titers raised a four fold or greater in 48.8% (61/125) in the control group and 35.1% (35/134) in the test group. There was a significant difference (p < 0.05) between the control and test groups. These results indicate that black tea extract is effective as a prophylactic agent against influenza infection.

[Two hepatocellular carcinoma patients with biloma after transarterial embolization with lipiodol (Lip-TAE) leading to occlusion of portal vein].

We presented two patients with post-Lip-TAE biloma resulting in portal occlusion, and reviewed 20 previous studies including our cases to investigate their clinical characteristics. Case 1. A 31-year-old woman suffered from an HCC located at the S8 segment, and had a superselective embolization of feeding arteries using 3 ml of Lip, 300 mg of CBDCA, and 40 mg of Epi-Adriamycin (Epi-ADM). Eleven weeks later, CT showed multiple cystic lesions, and the percutaneous transhepatic drainages of the lesions were established. At 21 weeks after Lip-TAE, we found occlusion of the right branch of portal vein on CT, but she recovered from this condition, and was discharged 1 year later. Case 2. A 62-year-old man was diagnosed as HCC located at S7-6 segments, and was infused with 3 ml of Lip, 150 mg of CBDCA, and 30 mg of Epi-ADM through a right hepatic artery. Ten weeks later, CT showed a cystic lesion in the S7-8 segments, occlusion of the right anterior segmental branch of the portal vein, and the same drainage was also established. Unfortunately, he died of liver failure 18 weeks later. In the literature, biloma after Lip-TAE occurred at 71.2 mean days, ranging from 7 to 180 days, a with remarkable increase in biliary tree-associated enzymes. Seven (35%) of 20 patients died of liver failure or sepsis during 3 weeks and 1 year, and 3 (60%) of 5 patients accompanied by occlusion of a certain portal branch frequently died. We consider that these patients need intensive care and should be under long follow-up.

[Efficacy of transarterial embolization combined with percutaneous ethanol injection therapy for recurrent hepatocellular carcinoma].

One hundred and eighty-nine patients with hepatocellular carcinoma (HCC) underwent hepatectomy since 1987 to 1992 in our institute. Recurrences were detected on residual liver in 84 patients up to December 1993. Sixty-seven of 84 patients were treated with re-resection (group-O, n = 11), transarterial embolization (TAE) combined with percutaneous ethanol injection therapy (PEIT) (group-TP, n = 13), TAE alone (group-T, n = 34) and intraarterial chemotherapy (group-IA, n = 9). Among these 67 cases, the efficacy of treatment for recurrences was investigated. There was no significant difference in age, positive ratio of hepatitis B virus, ICG R15 and percentage of underlying liver cirrhosis among the 4 groups. However, the frequency of patients with 2 nodules or less, was significantly higher in group-O than in other groups. Cumulative 1-, 2- and 3-year survival rates (%) were 88.9, 64.8 and 51.9 in group-O, 92.1, 55.4 and 55.4 in group-TP, 70.9, 49.6 and 31.0 in group-T, and 16.9, 0 and 0 in group-IA, respectively. The survival rate after recurrences in group-TP was higher than in group-IA and group-T, and almost equivalent to that of group-O. Either re-resection or TAE combined with PEIT might assure- a favorable prognosis in patients with recurrent HCC.

[Inhibition of the infectivity of influenza virus by black tea extract].

We determined whether black tea extract inhibits the infectivity of influenza virus to mice. When mice were inoculated intranasally with 10(5.3) PFU influenza viruses (10(1.3) LD50), their body weight decreased and all died within 10 days. Whereas, when mice were inoculated i.n. with the mixture of influenza viruses and 2% (w/w) black tea extract, 5 min after mixing, all mice showed normal body-weight increase and survived. Neutralizing antibody to influenza virus was not detected in nine of 10 survived mice. The results indicate that black tea extract at beverage concentration (2% w/w) inhibits almost completely the infectivity of influenza virus to mice and that in vivo reversion of the tea-inactivated influenza virus does not occur.

Inhibition of the infectivity of influenza virus by tea polyphenols.

(-)Epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) (1-10 microM) inhibited the infectivity of both influenza A virus and influenza B virus in Madin-Darby canine kidney (MDCK) cells in vitro. Study by electron microscope revealed that EGCg and TF3 (1 mM) agglutinated influenza viruses as well as did antibody, and that they prevented the viruses from adsorbing to MDCK cells. EGCg and TF3 more weakly inhibited adsorption of the viruses to MDCK cells. EGCg and TF3 (1-16 microM) also inhibited haemagglutination by influenza viruses. These findings suggest that tea polyphenols bind to the haemagglutinin of influenza virus, inhibit its adsorption to MDCK cells, and thus block its infectivity.

Bactericidal catechins damage the lipid bilayer.

The mode of antibacterial action of, the green tea (Camellia sinensis) extracts, (-)-epigallocatechin gallate (EGCg) and (-)-epicatechin (EC) was investigated. Strong bactericidal EGCg caused leakage of 5,6-carboxyfluorescein from phosphatidylcholine liposomes (PC), but EC with very weak bactericidal activity caused little damage to the membrane. Phosphatidylserine and dicetyl phosphate partially protected the membrane from EGCg-mediated damage when reconstituted into the liposome membrane with PC. EGCg, but not EC, caused strong aggregation and NPN-fluorescence quenching of PC-liposomes and these actions were markedly lowered in the presence of negatively charged lipids. These results show that bactericidal catechins primarily act on and damage bacterial membranes. The observation that Gram-negative bacteria are more resistant to bactericidal catechins than Gram-positive bacteria can be explained to some extent by the presence of negatively charged lipopolysaccharide.

Mitogenic activity of (-)epigallocatechin gallate on B-cells and investigation of its structure-function relationship.

(-)Epigallocatechin gallate (EGCg), the main constituent of green tea, strongly enhanced the direct plaque-forming cell (PFC) response to sheep red blood cells (SRBC) in vitro and showed strong mitogenic activity for mouse splenic B-cells but not for splenic T-cells and thymocytes. The enhancement of B-cell proliferation was not mediated by macrophages since their removal did not eliminate the activity. Among the derivatives of catechin examined, (+)catechin (C); (-)epicatechin (EC); (-)-epigallocatechin (EGC); (-)epicatechin gallate (ECg); (-)epigallocatechin gallate (EGCg); and theaflavin digallate (TF3), only the derivatives with the galloyl group (ECg, EGCg, and TF3) displayed significant enhancement of the spontaneous proliferation of B-cells. Structural analogs of the catechin and galloyl groups were also examined in the system. Gallic acid and tannic acid induced some enhancement, but rutin, pyrogallol and caffeine did not. The results indicate that the galloyl group on EGCg was responsible for enhancement. However, the basic conformations of the catechins are also important, because ECg, EGCg, TF3, gallic acid, and tannic acid had quite different potencies to induce B-cell proliferation.

Stimulation of human monocyte and polymorphonuclear cell iodination and interleukin-1 production by epigallocatechin gallate.

(-)Epigallocatechin gallate (EGCg) stimulated iodination of human peripheral blood monocytes, polymorphonuclear cells (PMN), and human promyelocytic leukemic HL-60 cells, dependent on time, dose, and temperature. However, EGCg did not affect iodination of nonadherent peripheral blood mononuclear cells, red blood cells, or 11 other cultured cell lines. Although various immunoregulators such as lipopolysaccharide (LPS), opsonized zymosan, 12-O-tetradecanoylphorbol-13-acetate (TPA) and tumor necrosis factor stimulated PMN iodination to varying degrees, their ability to stimulate monocyte iodination was much lower than that of EGCg. Washout experiments demonstrated that contact with EGCg for less than 60 min irreversibly stimulated PMN and monocyte iodination. EGCg also potently stimulated the production of interleukin-1-like factor by monocytes. The data suggest that EGCg is a strong in vitro stimulant of human phagocytes.

[Protective activity of tea and catechins against Bordetella pertussis].

We examined the bactericidal activity of tea and catechins against Bordetella pertussis. Green tea, black tea and coffee showed marked bactericidal activity at their concentrations in beverages, while pu-erh tea killed the bacteria in a moderate way. (-) Epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) showed also marked bactericidal activity. Green tea and black tea also effectively blocked the adhesion of B. pertussis to HeLa and CHO cells, whereas ECGg and TF3 could not. EGCg and TF3 markedly inactivated leuco-lymphocytosis promoting activity of pertussis toxin. Black tea showed slight but significant inactivation of the activity, whereas green tea showed no inactivation. These results suggest that green tea, black tea, EGCg and TF3 might act as prophylactic agents against pertussis infection.

[Antimicrobial and microbicidal activities of tea and catechins against Mycoplasma].

We examined tea extracts, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antimicrobial and microbicidal activities against Mycoplasma. Green tea and black tea showed antimicrobial activities against M. pneumoniae. At a concentration of 0.2% green tea and black tea showed microbicidal activities against M. pneumoniae and M. orale but not against M. salivarium. Extracts of pu-erh tea showed a slight microbicidal activity against M. pneumoniae and M. orale. EGCg purified from green tea and TF3 from black tea markedly showed microbicidal activities against M. pneumoniae. M. orale and M. salivarium. These results suggest that tea and catechins can be used as prophylactic agents against Mycoplasma pneumoniae infection.

The protective activity of tea catechins against experimental infection by Vibrio cholerae O1.

Tea catechins inhibited the fluid accumulation induced by cholera toxin in sealed adult mice. The catechins also reduced fluid accumulation by Vibrio cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea catechins may possess protective activity against V. cholerae O1.

[Antibacterial and bactericidal activities of tea extracts and catechins against methicillin resistant Staphylococcus aureus].

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antibacterial and bactericidal activities against methicillin resistant Staphylococcus aureus (MRSA) and food poisoning strains of S. aureus. Twenty percent tea extract (50 microliters), EGCg (63 micrograms) and TF3 (125 micrograms) added to one ml of culture medium each inhibited the growth of all strains of MRSA and food poisoning S. aureus tested. Tea extract showed also a bactericidal activity against MRSA even at the same concentration of as in ordinarily brewed tea. EGCg at a concentration of 250 micrograms/ml showed a bactericidal activity against MRSA but not against food poisoning S. aureus, but at 500 micrograms/ml reduced markedly the viable number within 48h. These results suggest that tea and catechin can be used as prophylactic agents against MRSA infection.