Intramitochondrial recruitment of endolysosomes mediates Smac degradation and constitutes a novel intrinsic apoptosis antagonizing function of XIAP E3 ligase.

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, BimEL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.

Naringin has an antiatherogenic effect with the inhibition of intercellular adhesion molecule-1 in hypercholesterolemic rabbits.

Naringin, a bioflavonoid found in citrus fruit peel, is known to have an antioxidative effect, but its effect on atherosclerosis has not been studied. This study evaluated the effect of naringin on blood lipid levels and aortic fatty streaks, and its action mechanism in hypercholesterolemic rabbits. Male New Zealand white rabbits were fed a 0.25% cholesterol diet and divided into an untreated group (n = 4), a naringin-treated group (n = 5; 500 mg/kg per day), and a lovastatin-treated group (n = 5; 20 mg/kg per day). After 8 weeks, blood was sampled and analyzed biochemically. Aorta and liver were harvested and examined histologically. Cholesterol level in rabbits fed the 0.25% cholesterol diet reached 17 times normal and decreased in the rabbits fed naringin and lovastatin, whose effects were not statistically significant (p > 0.05). However, both naringin and lovastatin effectively decreased the area of fatty streak in thoracic aorta on macroscopic analysis (p < 0.05) and significantly reduced subintimal foam cell infiltration on microscopic morphometry (p < 0.05). These foam cells were macrophages on immunohistochemical analysis. Naringin treatment inhibited hypercholesterolemia-induced intercellular adhesion molecule-1 (ICAM-1) expression on endothelial cells. Hypercholesterolemia caused fatty liver and elevation of liver enzymes, which was prevented by naringin but not by lovastatin. Naringin significantly reduced fatty streak formation and neointimal macrophage infiltration and also inhibited the expression of ICAM-1 in endothelial cells, suggesting that suppression of ICAM-1 contributed to the antiatherogenic effect. Naringin, unlike lovastatin, has a hepatoprotective action.

Analysis of mitochondrial DNA deletions in four chambers of failing human heart: hemodynamic stress, age, and disease are important factors.

Mitochondrial DNA (mtDNA) mutations are not only responsible for organ dysfunction due to inefficient energy production but also indicators of metabolic and functional stresses in the organ. To analyze the significance of deletion mutation in human myocardium, we screened the presence of two common deletions (7.4 kb from 8637-16084 nt, 5.0 kb from 8470-13477 nt) in four chambers using long-PCR, and using serial-dilution PCR, measured the amount of deleted mtDNA in normal heart (NL) of brain-dead victims of road accidents (n = 9, age = 10-59) and failing hearts (CHF) of patients who underwent heart transplantation (n = 24, age = 17-63). Frequency of both deletions was higher in ventricles (Vt) than in atria (At) (Vt:At = 25/33:12/33 for 7.4 kb, 19/33:6/33 for 5 kb) (p < 0.05), whereas it was the same in the right and left chambers. In ventricles, both deletions were more frequent among older persons (> 35 yrs) than in younger persons (< or = 35 yrs) (older:younger = 16/20:9/13 for 7.4 kb, 15/20:4/13 for 5 kb) (p < 0.05). In ventricles of failing heart, the 5-kb deletion was more frequent than in those of normal heart (CHF:NL = 17/24:2/9) (p < 0.05), whereas the 7.4-kb deletion was frequent both in failing and normal hearts (CHF:NL = 19/24:6/9). The association of mutation with aging or disease process observed in ventricles was not found in the atria. Although the amount of mutant mtDNA in the left ventricle tended to increase according to a disease process, it was small, at most 1.56% or 0.012% of total mtDNA for a 7.4- or 5-kb deletion, respectively. No deletion was found, however, in lymphocytes from any patient who underwent transplantation. In conclusion, deletion mutation of mtDNA is frequently, but in a small amount, found in the ventricle of older failing heart than in the atrium of younger normal heart. This suggests that hemodynamic stress, age, and disease are factors to induce mtDNA mutation that represents the indicator of stresses on the heart and might turn into a contributor of progressive heart failure under extreme conditions.

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms.

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.

Effect of red ginseng on blood pressure in patients with essential hypertension and white coat hypertension.

The objective of this study is to evaluate the changes of diurnal blood pressure pattern after 8 weeks of red ginseng medication (4.5 g/day) by 24 hour ambulatory blood pressure monitoring. In 26 subjects with essential hypertension, 24 hour mean systolic blood pressure decreased significantly (p = 0.03) while diastolic blood pressure only showed a tendency of decline (p = 0.17). The decrease in pressures were observed at daytime (8 A.M.-6 P.M.) and dawn (5 A.M.-7 A.M.). In 8 subjects with white coat hypertension, no significant blood pressure change was observed. We suggest that red ginseng might be useful as a relatively safe medication adjuvant to current antihypertensive medications.

Differentiation of Korean isolates of Entamoeba histolytica from Entamoeba dispar.

Cysts of Entamoeba histolytica are still found from humans in Korea, but not all of the cysts are known as pathogenic. The non-pathogenic strain is regarded as a different species, E. dispar. In this study, Korean isolates of conventional E. histolytica were subjected for the differentiation by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Human stools were screened by routine microscopic examination, and cyst or trophozoite positive stools were inoculated into Robinson media. The cultivated trophozoite positive stools were inoculated into Robinson media. The cultivated trophozoites were prepared for DNA extraction, and the DNAs were used for PCR with common primers of P1 gene. The PCR products were digested with 3 restriction enzymes and RFLP was observed. Also anti-sense primers containing the cleavage site of each restriction enzyme were designed for differentiation only by PCR. The PCR products of Korean isolates S9, S12, YS-6, and YS-27 were spliced by Taq I and Xmn I but not by Acc I, and the isolates S1, S3, S11, S15, S16, S17, S20, YS-17, and YS-44 were spliced by Acc I but not by Taq I and Xmn I. These RFLP pattern correlated well with PCR products by the species specific primers. The findings confirm that the Korean isolates S9, S12, YS-6, and YS-27 are E. histolytica and others are E. dispar. In Korea, most of the asymptomatic cyst carriers are infected by E. dispar, not by E. histolytica.