OBJECTIVETo determine the function of twin-arginine translocation system (Tat) and gene cluster in Vibrio strains and to analyze the homology of tat gene cluster among different Vibrio spp. strains based on N16961 and tatABC mutant strains N169-dtat.

METHODSDifferent serotypes of biotype strains of Vibrio spp. were selected to detect the transcription of 4 genes of Tat transport system and upstream ubi aarF gene and downstream cyt551 gene by the total RNA reverse transcription and homologicity of the gene cluster by sequencing analysis.

RESULTSOur results showed that the 4 genes of tat cluster (tatA, tatB, tatC, and tatE) were intragenic and co-transcribed. We found that ubi aarF gene could be co-transcribed with tatA, tatB, but not with tatC. The electron transport chain and energy metabolism-related genes, cytochrome C551 peroxidase gene, and 4 genes located at upstream of tatABC operon were not transcribed with tatABC. Although the co-transcription between ubi aarF and tatAB was blocked in N169-dtat strain, they were still transcribed separately. Homologous analysis of genes of tat cluster in different types of Vibrio cholerae showed that tat gene cluster was a very conservative.

CONCLUSIONThe ubi and aarF gene might be co-transcribed with genes of tat cluster in Vibrio cholerae, which and the close relationship showed that they might play a key function in Vibrio cholerae.

Arginine ; Bacterial Proteins ; Cytochrome c Group ; Membrane Transport Proteins ; Vibrio cholerae

OBJECTIVETo assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes.

METHODSHepatocytes isolated from tissue transglutaminase gene knock-out mice and rats were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by (14)C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.

RESULTSTTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.

CONCLUSIONSIncreased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

Animals ; Apoptosis ; Cell Nucleus ; metabolism ; Cytochrome c Group ; metabolism ; Hepatocytes ; cytology ; metabolism ; Male ; Mice ; Rats ; Signal Transduction ; Transglutaminases ; metabolism

Impairment of neuronal mitochondria following hypoxia of brain not only result in nerve cell's energy-deprivation and dysfunction, mitochondria also play key roles in apoptosis of neurons. A central step being the release of cytochrome c (cyt c) across the outer mitochondrial membrane into the cytoplasm through opening of the mitochondrial permeability transition pore. Releasing of cytochrome c induce to downstream consequences of specific caspase activation. The antiapoptotic and proapoptotic members of the Bcl-2 family regulate mitochondrial activities relevant to apoptotic signaling by influencing the realaseing of cyt c.
Apoptosis ; Caspases/metabolism* ; Cytochrome c Group/metabolism* ; Humans ; Hypoxia, Brain/pathology* ; Membrane Proteins/metabolism* ; Mitochondria/metabolism* ; Neurons/pathology* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Signal Transduction

Although widely studied, the natural diversity of the hard tick is not well known. In this study, we collected 194 sequences from 67 species, covering 7 genera of hard tick. The 5′ region of the mitochondrial cytochrome c oxidase subunit 1 region (586 bp) has been used to investigate intra- and inter-species variation and the phylogenetic tree of neighbor joining method has been used for assessment. As a result, by comparing the K2P-distance of intra- and interspecies, 30 samples (15.2%) shown that interspecies distance was larger than the minimum interspecfic distance. From the phylogenetic analysis, 86.8% (49) of the species were identified correctly at the genus level. On deeper analysis on these species suggested the possibility of presence cryptic species. Therefore, further work is required to delineate species boundaries and to develop a more complete understanding of hard tick diversity over larger scale.
Cytochromes c ; Cytochromes ; Electron Transport Complex IV ; Ixodidae ; Methods ; Trees

Cytochromes c covalently bind their heme prosthetic groups through thioether bonds between the vinyl groups of the heme and the thiols of a CXXCH motif within the protein. In Gram-negative bacteria, this process is catalyzed by the Ccm (cytochrome c maturation) proteins, also called System I. The Ccm proteins are found in the bacterial inner membrane, but some (CcmE, CcmG, CcmH, and CcmI) also have soluble functional domains on the periplasmic face of the membrane. Elucidation of the mechanisms involved in the transport and relay of heme and the apocytochrome from the bacterial cytosol into the periplasm, and their subsequent reaction, has proved challenging due to the fact that most of the proteins involved are membrane-associated, but recent progress in understanding some key components has thrown up some surprises. In this Review, we discuss advances in our understanding of this process arising from a substrate's point of view and from recent structural information about individual components.
Cytochromes c ; chemistry ; metabolism ; Models, Biological

OBJECTIVETo investigate the effects of anti-apoptosis gene (bcl-X(L)), cytochrome c and caspase-3 activity on chemoresistance in cisplatin-resistant human ovarian cancer cell lines (A2780/DDP, COC1/DDP).

METHODSThe expression of bcl-X(L) cisplatin treated cytochrome c and caspase-3 activity were monitored by RT-PCR and Western blot in cisplatin-resistant (A2780/DDP, COC1/DDP) and cisplatin-sensitive (A2780, COC1) cell lines. The apoptotic rates of A2780, COC1, A2780/DDP and COC1/DDP were detected with flow cytometry after having been treated by cisplatin.

RESULTSThe expression of bcl-X(L) in A2780/DDP and COC1/DDP was significantly higher than that in A2780 and COC1 cells, whereas the expression of cytochrome c, caspase-3 activity and apoptotic rates of A2780/DDP and COC1/DDP were significantly reduced more than those of A2780 and COC1 after having been treated by cisplatin (P < 0.05).

CONCLUSIONThe overexpression of anti-apoptotic gene bcl-X(L), which downregulates cytochrome c and decreases caspase-3 activity, may be related to cisplatin-resistance in human ovarian cancer cell lines.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Caspase 3 ; Caspases ; metabolism ; Cisplatin ; pharmacology ; Cytochrome c Group ; metabolism ; Drug Resistance, Multiple ; physiology ; Drug Resistance, Neoplasm ; physiology ; Female ; Humans ; Ovarian Neoplasms ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tumor Cells, Cultured ; bcl-X Protein

Herba houttuyniae has been used as a constituent of herval medicine prescriptions for the treatment of inflammation, cancer, and other diseases. In the present study, we investigated the cellular effects of herba houttuyniae extract (HHE) and the signal pathways of HHE-induced apoptosis in HL-60 human promyelocytic leukemia cell line. HHE treatment caused apoptosis of cells as evidenced by discontinuous fragmentation of DNA, the loss of mitochondrial membrane potential, release of mitochondrial cytochrome c into the cytosol, activation of procaspase-9 and caspase-3, and proteolytic cleavage of poly(ADP-ribose) polymerase. Pretreatment of Ac-DEVD-CHO, caspase-3 specific inhibitor, or cyclosporin A, a mitochondrial permeability transition inhibitor, completely abolished HHE-induced DNA fragmentation. Together, these results suggest that HHE possibly causes mitochondrial damage leading to cytochrome c release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HL-60 cells.
Apoptosis/*drug effects ; Caspases/*metabolism ; Cytochrome c Group/*metabolism ; Enzyme Activation/drug effects ; HL-60 Cells ; Human ; Medicine, Oriental Traditional ; Membrane Potentials/drug effects ; Mitochondria/*drug effects ; Plant Extracts/*pharmacology ; Plants, Medicinal/*chemistry

The permeability of mitochondrial outer membrane (MOM) is regulated by the proteins of the Bcl-2 family via their interactions at the membrane. While pro-apoptotic Bax protein promotes MOM permeabilization (MOMP) releasing cytochrome c after activation by BH3-only protein, anti-apoptotic Bcl-2 protein protects MOM. However both Bax and Bcl-2 can form pores in model membranes. Unlike Bax pore that has been extensively studied and reported to be directly linked to MOMP, Bcl-2 pore is much less known; thus we investigated the pore-forming property of recombinant Bcl-2 lacking the C-terminal transmembrane sequence (Bcl-2deltaTM) in liposomal membranes of MOM lipids. We found that: (1) Bcl-2 formed pores at acidic pH that induced the association of Bcl-2 with liposome; (2) Bcl-2 pore size was dependent on Bcl-2 concentration, suggesting that oligomerization is involved in Bcl-2 pore formation; (3) Unlike Bax pore that could release large molecules up to 2 mega-Da, Bcl-2 pore was smaller and could only release the molecules of a few kilo-Da. Therefore, Bcl-2 and Bax may form different size pores in MOM, and while the large pore formed by Bax may release cytochrome c during apoptosis, the small pore formed by Bcl-2 may maintain the normal MOM permeability.
BH3 Interacting Domain Death Agonist Protein ; metabolism ; Cell Membrane Permeability ; Cytochrome c Group ; metabolism ; Humans ; Hydrogen-Ion Concentration ; Liposomes ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism

Fumaric acid esters (FAE), mainly dimethylfumarate (DMF), have been shown to be highly efficacious in the treatment of psoriasis. Among the potential side effects of FAE therapy, lymphocytopenia is sometimes observed. In order to address the question whether FAE may interfere with systems of the innate defense, the modulatory role of FAE on the generation of superoxide-anion by human monocytes and neutrophils was studied by measuring the reduction of cytochrome c. Various concentrations of DMF and its metabolite methylhydrogenfumarate (MHF) were used to observe their modulatory effect on superoxide-anion generation by monocytes and neutrophils in response to bacteria (S. aureus and E. coli) and candida (C. albicans). Dexamethasone (DXM, 1 x 10(-7) mol x L(-1)) was also studied at the same time. We found that DXM significantly inhibited superoxide-anion generation from monocytes in response to bacteria and C. albicans, whereas DMF and MHF (10-20 microg x mL(-1)) significantly increased the production of superoxide-anion in monocytes in response to the above mentioned bacteria. DXM, DMF and MHF did not affect superoxide-anion generation of neutrophils. Our data indicate that DMF and MHF enhance superoxide-anion generation in human monocytes as one of the important mechanisms of innate defense against microorganisms.
Candida albicans ; immunology ; Cells, Cultured ; Cytochrome c Group ; metabolism ; Dermatologic Agents ; pharmacology ; Dimethyl Fumarate ; Escherichia coli ; immunology ; Fumarates ; pharmacology ; Humans ; Phagocytes ; metabolism ; Staphylococcus aureus ; immunology ; Superoxides ; metabolism ; Zymosan ; immunology

BACKGROUND: Microinjectors have been used for cell biology and development, and are useful for the study of cellular morphologic changes with response to the external milieu and intracellularly injected molecules. METHODS: This study was performed to confirm the apoptotic changes induced by intracytoplasmic microinjection of cytochrome c (5 mg/mL) to mouse 3T3 fibroblasts with and without pretreatment of Ac-DEVD-CHO (100 mol/mL), and BSA (bovine serum albumin, 5 mg/mL) as a control, and evaluate the usefulness of microinjection as a method to study apoptosis pathways. RESULTS: Mild focal cytoplasmic fragmentation was seen in the cells microinjected with cytochrome c as early as 10 min after the injection. Apoptotic morphology with apoptotic body formation was observed at 60 min after the injection, and then new apoptotic change of the injected cells was not seen. Cytochrome c-injected cells showed about 31% of apoptotic cells of the total injected cells 50-60 min after the injection. BSA-injected cells did not show apoptosis morphology at 50-60 min after the injections. Caspase-3 inhibitor, Ac-DEVD-CHO-treated cells with cytochrome c microinjection exhibited lower apoptosis indices (average apoptosis index; 11.5+/-8.6%) than non-treated cells of the inhibitor (average apoptosis index; 11.5+/-8.6%). CONCLUSIONS: It was observed that intracellular microinjection of cytochromic c induced apoptosis which was inhibited by Ac-DEVD-CHO, although apoptotic cells were so easily detached that further study could not be performed. However it is thought that microinjection should be a method to study apoptosis and signal transduction with the molecular biological techniques currently available.
Animals ; Apoptosis* ; Caspase 3 ; Cytochromes c* ; Cytochromes* ; Cytoplasm ; Fibroblasts* ; Mice* ; Microinjections* ; Serum Albumin ; Signal Transduction