• Bulletin of the Korean Chemical Society
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• 제27권9호
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• pp.1335-1340
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• 2006

It has been found that a number of diseases are associated with mutations in the mitochondrial DNA. Therapeutic gene delivery to mitochondria has been suggested as a clinical option for these diseases. In this study, we developed a gene carrier to mitochondria by the conjugation of mitochondrial leader peptide (LP) to polyethylenimine (PEI). Mitochondrial LP conjugated PEI (PEI-LP) was synthesized with low molecular weight PEI (2,000 Da, PEI2K). Gel retardation assay showed that PEI2K-LP formed complexes at a 1.0/1 weight ratio. In addition, PEI2K-LP protected DNA from the enzymatic degradation for at least 60 min, while naked DNA was completely degraded within 20 min. PEI2K-LP was compared with LP conjugated high molecular weight PEI (25,000 Da, PEI25K) in terms of toxicity and delivery efficiency. MTT assay showed that PEI2K-LP had much lower cytotoxicity than PEI25K-LP to 293 cells. In addition, cell-free DNA delivery assay showed that PEI2K-LP delivered more DNA to mitochondria at a 1.8/1 weight ratio than naked DNA or PEI. This result suggests that PEI2K-LP may be useful for the development of mitochondrial gene therapy system with lower cytotoxicity.

• Applied Microscopy
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• 제24권3호
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• pp.55-66
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• 1994

This study was carried out to investigate the histological changes of sperm cells in testis, obtained from 100 of 3-year-old male rainbow trout (Oncorhynchus mykiss) collected and analysed from March in 1992 to February in 1993. Especially, the ultrastructural changes of spermatogonia, primary and secondary spermatocytes, spermatids, and spermatozoa were examined to describe the reproductive cycles of this species. The results obtained in this study were as follows: The ultrastructures of the gonadotrophs largely parallel the cyclical changes in the testes. Each nest of cells belongs to one spermatogenetic stage, although nests at different stages can be found within the one lobule. At first keterochromatin is dispersed and then is condensed. In mature gamete, the nucleus is dense and homogeneous. The nuclear membrane appeared at the beginning of differentiation. In spermatogonia, Sertoli cells are located at the periphery of their cytoplasm. In the primary spermatocytes, the small mitochondria are abundant over the outer cytoplasm. During cell differentiation, the cytoplasm decreases and the nucleus increases. In spermatids, the protein masses moved towards the posterior part of the nucleus. In late spermatids, the two large mitochondria are located over the cytoplasm. In spermatozoa, two spheroidal mitochondria (about 145nm long) are situated in parallel between the nucleus and the axoneme. Spermatozoa mitochondria are assembled into an organized sheath surrounding the outer dense fibres and axoneme of the flagellar midpiece. The two centrioles are quite separate and the central pair and sheath complex of the flagellum is inserted into the base of the distal centriole.

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.141-148
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• 1998

돌고기 Pungtungia herzi 정자의 미세구조를 주사 및 투과 전자현미경적 방법으로 연구하였다. 정자는 구형의 두부, 짧은 중편 및 꼬리로 구성되어 있으며 비교적 그 구조가 단순하고 그 길이는 약 37.4$mu extrm{m}$이었다. 정자의 미세구조에서 핵에 대한 접선 방향으로의 편모 배열, 짧은 핵 그리고 미토콘드리아의 비대칭적 배열은 잉어과 정자의 공통된 특징과 마찬가지로 나타났으며, 대부분의 경골어류에서와 같이 첨체를 가지고 있지 않았다. 반면 돌고기 정자에서는 핵막의 구조와 미토콘드리아에서 현저한 특징을 나타내었다. 핵막은 매우 심하게 파동되어 있으며 얕은 핵와내의 기부 및 말단중심립은 약 130$^{\circ}$의 각도로 배열되어 있었다. 미토콘드리아는 제 1 정모세포기에 5개 이상 관찰되었으나 성숙한 정자에서는 하나의 형태로 융합되어 있었다. 이러한 양상은 융합되지 않은 다른 잉어류 정자에서의 미토콘드리아와 비교할 때 현저한 차이가 있었다. 정자의 계통 진화적 견지에서, 돌고기에서와 같이 융합된 미토콘드리아는 융합되지 않고 분리되어 있는 미토콘드리아에 비해 원거리 형질(apomorphic character)로 간주된다. 한 개의 미토콘드리아를 가지는 정자는 현재까지 Rhodeus 이외의 다른 잉어류 정자에서는 보고된 바 없었다.

• 생명과학회지
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• 제10권5호
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• pp.496-503
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• 2000

This study was designed to investigate the effects of silkworm (Bombyx mori L.) powder on oxidative stress and membrane fluidity in liver membranes of rats. Sprague-Dawley (SD) male rats (160±10 g) were fed basic diet (control group), and experimental diets (SWP-200 and SWP-400 groups) added 200 and 400 mg/kg BW/day for 6 weeks. A significant differences between liver mitochondria and microsomes of SWP-200 and SWP-400 groups could not be obtained. Membrane fluidities were dose-dependently increased (14.8% and 28.5%, 20.0% and 29.9%) in liver mitochondria and microsomes of SWP-200 and SWP-400 groups compared with control group. Basal oxygen radicals (BOR) in liver mitochondria and mocrosomes were significantly inhibited (15.2% and 21.7%, 12.6% and 18.6%, respectively) by SWP-200 and SWP-400 groups compared with control group. Induced oxygen radicals (IOR) in liver microsomes were significantly inhibited (15.5% and 16.1%, respectively) by SWP-200 and SWP-400 groups compared with control group, but IOR in liver mitochondria was significantly inhibited about 12.0% by SWP-400 group only compared with control group. Lipid peroxide (LPO) levels were significantly decreased (14.4% and 9.1%, respectively) in liver mitochondria and microsomes of SWP-400 group only compared with control group. Oxidized protein (OP) levels were remarkably decreased about 12.7% and 16.3% in liver microsomes only of SWP-200 and SWP-400 groups, but significant difference between liver motochondria could not obtained. These results suggest that administration of SWP may play an effective role in a attenuating a oxidative stress and increasing a membrane fluidity in liver membranes.

• 생명과학회지
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• 제10권5호
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• pp.511-518
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• 2000

This study was designed to investigate the effects of silk fibroin powder (SFP : Mw 500) on oxidative stress and membrane fluidity in brain membranes of rats. Sprague-Dawley (SD) male rats (160$\pm$10 g) were fed basic diet (control group), and experimental diets (SFP-2.5 and SFP-5.0 groups) added 2.5 and 5.0 g/kg BW/day for 6 weeks. Cholesterol level was significantly decreased about 8.0% in brain microsomes of SFP-5.0 group only compared with control group. Membrane fluidities were significantly increased (12.9% and 15.2%, respectively) in brain microsomes of SFP-2.5 and SFP-5.0 groups, but significant difference between in brain mitochondria of these two groups could be not obtained. Basal oxygen radicals (BOR) in brain mitochondria and microsomes were significantly ingibited (10.4%, and 24.0%, 7.9% and 14.9%, respectively) by SFP-2.5 and SFP-5.0 groups compared with control group. Induced oxygen radicals (IOR) in brain mitochondria and microsomes were significantly inhibited (11.8% and 14.1%, respectively) by SFP-5.0 groups compared with control group compared with control group. Lipid peroxide (LPO) levels were dose-dependently decreased (12.9% and 21.9%, 13.2% and 22.5%, respectively) in brain mitochondria and microsomes of SFP-2.5 and SFP-5.0 groups compared with control group. Oxidized protein (OP) levels were significantly decreased (15.7% and 17.1%, 16.7% and 15.7%, respectively) in brain mitochondria and microsomes of SFP-2.5 and SFP-2.5 and SFP-5.0 groups compared with control group. These results suggest that administration of SFP may play an effective role in a attenuating a oxidative stress and increasing a membrane fluidity in brain membranes.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.141-144
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• 2005

This experiment was to investigate whether the mitochondria function assessment can be used for the prediction of sperm fertility through examining the correlation between mitochondria fluoromicroscopic frequency of frozen-thawed eight Hanwoo bull semen using rhodamine123 (R123) and in vitro embryo development following fertilization. Individual sperm were stained in 5 ${\mu}g/mL$ R123-added calcium-free Sp-TALP for 30 min at 0 h, 6 h, 12 h and 24 h after thawing and examined their mid-piece under an epifluorescence microscope using 495 nm excitation filter (x1,000). Three replications were taken, and at least 300 sperm per individual were examined. When semen samples were separated into two groups (good and poor) by sperm motility and fluorescent frequencies at just after thawing, average fluorescent frequencies were remarkably reduced as time going (0 h; $53.29{\~}72.94\%$, 6 h; $21.40{\~}58.90\%$, 12 h; $8.26{\~}25.93\%$, 24 h; $1.00{\~}13.78\%$, irrespective of selected group, and there were no differences at 6 h or 12 h after thawing between selected groups but indicated significant difference at 24 h after thawing (p<0.05). In vitro fertilization rates in good and poor groups ranging $70.8{\~}77.8\%$ and $52.1{\~}84.5\%$, respectively, were not significantly different. However, in vitro development rates of the same groups ranging $25.7{\~}40.0\%$ and $12.9{\~}1.8\%$, respectively, were significant different (p<0.05). These results demonstrate that mitochondria fluoromicroscopic assessment of frozen-thawed bovine sperm may be used as a criterion to select more fertile sperm.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1142-1148
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• 2020

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans, a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this study, we identified and functionally characterized Mrs3/4, a homolog of the Saccharomyces cerevisiae mitochondrial iron transporter, in C. neoformans var. grubii. A strain expressing an Mrs3/4-GFP fusion protein was generated, and the mitochondrial localization of the fusion protein was confirmed. Moreover, a mutant lacking the MRS3/4 gene was constructed; this mutant displayed significantly reduced mitochondrial iron and cellular heme accumulation. In addition, impaired mitochondrial iron-sulfur cluster metabolism and altered expression of genes required for iron uptake at the plasma membrane were observed in the mrs3/4 mutant, suggesting that Mrs3/4 is involved in iron import and metabolism in the mitochondria of C. neoformans. Using a murine model of cryptococcosis, we demonstrated that an mrs3/4 mutant is defective in survival and virulence. Taken together, our study suggests that Mrs3/4 is responsible for iron import in mitochondria and reveals a link between mitochondrial iron metabolism and the virulence of C. neoformans.

• Journal of Korean Neurosurgical Society
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• 제29권10호
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• pp.1283-1288
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• 2000

Objectives : The subcellular locations of Bad, Bid, Bax and Bcl-XL change during apoptosis and this change is important for the regulation of cell death. The purpose this study was to elucidate binding of Bad with Bcl-XL in vivo Methods : We mads Bad with Green Fluorescent Protein(GFP) using PCR method. We transfected and overexpressed GFP-Bad with or without Bcl-XL cotransfection in living COS-7 cell. Results : Bad and Bcl- XL bind one another in healthy living cells and this association controled mitochondrial docking. In the absence of Bad-XL, Bad was mainly cytosolic and partially bound to mitochondria. Upon coexpression of Bad and Bcl-XL, most of Bad translocated to mitochondria. These should suggest that Bad binds to the mitochondrial and cytoplasmic forms of Bcl-XL and Bad bound to cytoplasmic Bcl-XL translocates to mitochondria. These in vivo findings confirm that Bad make a complexes with Bcl- XL and cause mitochondrial translocation of Bad-Bcl-XL complex.

• The Korean Journal of Physiology and Pharmacology
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• 제9권1호
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• pp.29-35
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• 2005

To prove the buffering contribution of mitochondria to the increase of intracellular $Ca^{2+}$ level ($[Ca^{2+}]_i$) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial $Ca^{2+}$ entry and $Ca^{2+}$ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM ($R_{340/380}$) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh ($10{\mu}M$) increased $R_{340/380}$ by $1.1{\pm}0.15$ ($mean{\pm}S.E.$, n=6). When the external $Na^+$ was totally replaced by $NMDG^+$, $R_{340/380}$ was increased by $1.19{\pm}0.17$ in a reversible manner (n=27). $NMDG^+$-induced $[Ca^{2+}]_i$ increase was followed by oscillatory decay after $[Ca^{2+}]_i$ reached the peak level. The increase of $[Ca^{2+}]_i$ by $NMDG^+$ was completely suppressed by replacement with $Cs^+$. When $1{\mu}M$ CCCP was applied to bath solution, the ratio of $[Ca^{2+}]_i$ was increased by $0.4{\pm}0.06$ (n=31). When $1{\mu}M$ CCCP was used for pretreatment before application of $NMDG^+$, oscillatory decay of $[Ca^{2+}]_i$ by $NMDG^+$ was significantly inhibited compared to the control (p＜0.05). In addition, $NMDG^+-induced$ increase of $[Ca^{2+}]_i$ was highly enhanced by pretreatment with $2{\mu}M$ CCCP by $320{\pm}93.7$%, compared to the control ($mean{\pm}S.E.$, n=12). From these results, it is concluded that mitochondria might have buffering contribution to the $[Ca^{2+}]_i$ increase through regulation of the background NSCC in RAECs.

• 약학회지
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• 제48권6호
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• pp.352-357
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• 2004

Atrial myocytes have two functionally separate groups of ryanodine receptors (RyRs): those at the periphery colocalized with L-type $Ca^{2+}$channels (DHPRS) and those a t the cell interior not associated with DHPRs. $Ca^{2+}$ current ($I_{ca}$) directly gates peripheral RyRs on action potential and the subsequent peripheral $Ca^{2+}$ release propagates into the center of atrial myocytes. The mechanisms that regulate the $Ca^{2+}$+ propagation wave remain Poorly understood. Using 2-D confocal$Ca^{2+}$ imaging, we examined the role of inositol 1,4,5-trisphosphate receptor (IP $_3R$) and mitochondria on ($I_{ca}$)- gated local $Ca^{2+}$ signaling in rat atrial myocytes. Blockade of IP $_3R$ by xestospongin C (XeC) partially suppressed the magnitudes of I ca-gated central and peripheral $Ca^{2+}$ releases with no effect on $I_{ca}$. Mitochondrial staining revealed that mitochondria were aligned with ${\thickapprox}2-{\mu}m$ separations in the entire cytoplasm of ventricular and atrial myocytes. Membrane depolarization induced rapid mitochondrial $Ca^{2+}$ rise and decay in the cell periphery with slower rise in the center, suggesting that mitochondria may immediately uptake cytosolic $Ca^{2+}$, released from the peripheral SR on depolarization, and re-release the $Ca^{2+}$ into the cytosol to activate neighboring central RyRs. Our data suggest that the activation of IP $_3R$ and mitochondrial $Ca^{2+}$ handing on action potential may serve as a cofactor for the $Ca^{2+}$ propagation from the DHPR-coupled RyRs to the DHPR-uncoupled RyRs with large gaps between them.