Analysis of twin-arginine translocation system gene homology and transcription in Vibrio species / 中华预防医学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Different 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.</p><p><b>RESULTS</b>Our 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.</p><p><b>CONCLUSION</b>The 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.</p>

Sheep gastrointestinal helminthiasis in the Sertão region of Paraíba State, Northeastern Brazil: prevalence and risk factors / Helmintoses gastrintestinais de ovinos no Sertão do Estado da Paraíba, Nordeste do Brasil: prevalência e fatores de riscos

In this study, we aimed to establish the prevalence and risk factors relating to gastrointestinal helminthiasis, and to characterize the sanitary management practiced among sheep herds in the Sertão region of the state of Paraíba, northeastern Brazil, based on factors that condition the ways of controlling these parasites in these herds. The research was carried out between April and July 2012. We visited 54 farms, where fecal and blood samples were individually collected from 465 animals. On each farm, a questionnaire was applied to gather information on variables relating to potential risk factors. The prevalence of sheep gastrointestinal helminthiasis in the region was 75.9%. At least one animal tested positive for this helminthiasis on 53 (98.1%) of the 54 farms evaluated. The eggs per gram of feces (EPG) analysis showed the following infection burdens: 51.8% with mild infection, 27.1% moderate infection, 9.9% heavy infection and 11.2% fatal infection. Among the sheep farms visited, anthelmintics were used on 81.5% (p <0.05). The most relevant risk factor in this study was the farm area, because it defines the area available for grazing animals. Properties with many animals and little pasture area, which are the most abundant type in the Sertão region of Paraíba, tend to have high prevalence of gastrointestinal helminthiasis, because the animals are more prone to reinfection. The Sertão region of Paraíba presents high prevalence of gastrointestinal helminthiasis among sheep, and the farm area is the most relevant risk factor for the development of these parasites.

Objetivou-se determinar a prevalência e os fatores de risco para as helmintoses gastrintestinais, caracterizando o manejo sanitário sob fatores condicionantes das formas de controle dessas parasitoses em rebanhos de ovinos da região do Sertão da Paraíba. A pesquisa foi desenvolvida no período de abril a julho de 2012. Foram visitadas propriedades, utilizando-se 465 animais, sendo coletadas individualmente amostras de fezes e sangue durante as visitas. Em cada propriedade, foi aplicado questionário para a coleta de informações acerca de variáveis que atuariam como possíveis fatores de risco. Observou-se que a prevalência das helmintoses gastrintestinais de ovinos na região do Sertão da Paraíba foi de 75,9%. Pelo menos um animal foi positivo para essas helmintoses, em 53 (98,1%) das 54 propriedades avaliadas. A análise de OPG (Ovos Por Gramas de Fezes) demonstrou que 51,8% dos animais apresentaram infecção leve, 27,1% infecção moderada, 9,9% infecção pesada e 11,2% infecção fatal. A utilização de anti-helmínticos ocorreu em 81,5% das propriedades (p <0,05). O fator de risco mais relevante neste estudo foi a área da propriedade, porque delimita a área de pastejo do animal. Propriedades com muitos animais e pouca área de pastejo, que são as mais abundantes no Sertão da Paraíba, tendem a apresentar alta prevalência de helmintoses gastrintestinais, pois os animais estão mais propensos à reinfecção. A região do Sertão da Paraíba apresenta uma elevada prevalência de helmintoses gastrintestinais em ovinos, e a área das propriedades é o fator de risco mais relevante para o desenvolvimento dessas parasitoses.

Investigation of cytocrom c oxidase gene subunits expression on the Multiple sclerosis.

INTRODUCTION: Multiple sclerosis (MS) is an autoimmune inflamatory disease, which affects the (Central Nervous System) and leads to the destruction of myelin and atrophy of the axons. Genetic factors, in addition to environmental ones, seem to play a role in MS. Numerous studies have reported mitochondrial defects including a reduction in cytochrome c oxidase (COX) complex function related to the reduction of mitochondrial genes expression in the cortex tissue of patients with MS have been reported. MATERIALS AND METHODS: This study aimed to assess COX5B and COX2 genes expression in MS patients and compare it with normal subjects. We determine expression levels of genes COX5B and COX2, and also gene reference ß-actine using real–time polymerase chain reaction (RT-PCR) method. Data were obtained and obtained and standardized with the gene reference and were analyzed using independent sample t-test with SPSS and Excel programs. RESULT AND DISCUSSION: The resultshowed COX5B gene expression reduced significant in MS patients compared to normal subjects (P < −0.05) whereas, there was no significant difference in the COX2 gene expression between normal subjects and patients. Thus, it can be claimed that down-regulation of mitochondrial electron transport chain genes supported the hypothesis that hypoxia-like tissue injury in MS may be due to mitochondrial genes, different expression impairment.

Antitumor effect of capsaicin on colorectal carcinoma xenograft in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the effect of capsaicin on nude mice xenografted with colorectal carcinoma cells, and to explore its mechanism of action.</p><p><b>METHODS</b>A nude mouse model of colorectal cancer was established by subcutaneous inoculation of human colorectal carcinoma HT-29 cells. Terminal deoxynucleotidyl transferase-mediated nicked labeling assay (TUNEL) was undertaken to detect the cell proliferation and apoptosis in the xenograft tissue in nude mice. Immunohistochemical (IHC) staining and Western blot were used to detect the expression of HSP27, Cyt-C and active caspase-3.</p><p><b>RESULTS</b>The tumor growth of the groups C10 and C20 was significantly slower than that of the group NS. The integrated optical density (IOD) of both the group C5 (2532.14 ± 578.11) and group C10 (6364.03 ± 1137.98) was significantly higher than that of the group NS (760.12 ± 238.05), (P < 0.05). The integrated optical density (IOD) of the group C20 was (15743.96 ± 1855.95), significantly higher than that of the groups C10, C5 and NS (all were P < 0.01). Immunohistochemistry showed that the cytoplasmic expression of HSP27 was strongly positive in the group NS, and significantly reduced with the increasing dose of capsaicin in the treated groups. The expression of active caspase-3 and Cyt-C in the group NS was weakly positive, and was significantly increased with the increasing dose of capsaicin in the groups C5 and C10 (P < 0.05), and the expression of active caspase-3 and Cyt-C of the group C20 was significantly higher than that of the groups C5, C10 and NS (P < 0.01). Western blot analysis showed that both the expressions of HSP27 of the group C5 (0.73 ± 0.05) and the group C10 (0.41 ± 0.03) were significantly lower than that of the group NS (P < 0.05). The expression of HSP27 of the group C20 (0.22 ± 0.06) was significantly lower than that of the groups C5, C10 and NS (P < 0.01). The expressions of active-caspase-3 and Cyt-C in the group C5 were (2.57 ± 0.34) and (2.03 ± 0.38), significantly higher than those of the group NS (P < 0.05). The expressions of active-caspase-3 and Cyt-C in the group C10 were (4.23 ± 0.45) and (3.13 ± 0.44), also significantly higher than those of the group NS (P < 0.05). The expressions of active-caspase-3 and Cyt-C in the group C20 were (5.78 ± 0.48) and (4.92 ± 0.52), significantly higher than those of the group C5, C10 and NS (P < 0.01). TUNEL analysis showed that there was a significant difference of cell apoptosis in comparison of each two groups. The higher dose of capsaicin was used, the more apoptosis was observed.</p><p><b>CONCLUSIONS</b>Capsaicin can significantly inhibit the tumor growth and induce cell apoptosis in the colorectal carcinoma xenograft in nude mice. Its mechanism of action is possibly related with the down-regulation of HSP27 expression and up-regulation of expression of active caspase-3 and Cyt-C in the colorectal carcinoma xenograft in nude mice.</p>

The cytosolic domain of Bcl-2 forms small pores in model mitochondrial outer membrane after acidic pH-induced membrane association / 生物医学工程学杂志

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.

Enhanced cytochrome C oxidase activity in WBC of COPD patients

Chronic obstructive pulmonary disease [COPD] is characterized by decreased expiratory flow rates, increased pulmonary resistance and hyperinflation. Cytochrome C Oxidase [COX] as a key oxidative enzyme modulates oxygen uptake and catalyzes the oxidation of reduced cytochrome C by molecular oxygen. In vitro studies indicate that the activity of COX can be directly regulated by the presence of molecular oxygen. Thus, a better understanding of the role of COX in patients with COPD can provide an important link between the availability of oxygen to tissues and the regulation of oxygen uptake and energy production in these patients. We studied 42 COPD patients [36 males, 6 females] with clinically stable conditions and 50 [42 males, 8 females] healthy sedentary volunteers of similar age. Whole blood was collected by venipuncture in sodium citrate tubes and WBCs were separated by Ficoll according to standard protocol and lysed with microtube pestle homogenizer. The homogenates were centrifuged and the supernatants were used as a cell extract for COX activity determination. Aliquots of this were assayed for total protein content and COX activity. Analysis of COX activity was performed using COX assay kit. Absolute specific COX activity was normalized for total protein. Relative activities were determined by dividing absolute specific COX activity on absolute specific citrate synthase activity. Mitochondrial COX activity and specific activity [absolute and relative] significantly increased in WBCs of patients with COPD in comparison with control samples [p< 0.05] These results indicated that the activity of COX was increased in WBCs of patients with COPD but whether this is a primary or secondary change relevant to hypoxic condition in these patients is not clear and needs further investigation.

Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors / 대한진단검사의학회지

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.

Modulatory effect of fumaric acid esters on superoxide-anion generation in human phagocytes / 药学学报

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.

Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK-stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.

Mitochondrial apoptotic signaling pathway in neurons following brain injury induced by hypoxia / 法医学杂志

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.

Effects of nuclear translocation of tissue transglutaminase and the release of cytochrome C on hepatocyte apoptosis / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Hepatocytes 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.</p><p><b>RESULTS</b>TTG 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.</p><p><b>CONCLUSIONS</b>Increased 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.</p>

Herba houttuyniae extract induces apoptotic death of human promyelocytic leukemia cells via caspase activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release

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.

Relationship between cytochrome c-mediated caspase-3 activity and chemoresistance in cisplatin-resistant human ovarian cancer cell lines / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To 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).</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>The 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.</p>

The mechanism of STI571 inducing apoptosis of K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of STI571 inducing apoptosis of K562 cells which express P210(BCR/ABL).</p><p><b>METHODS</b>Apoptosis was analyzed by Annexin-V/PI, DioC6 [3] staining, DCFH-DA staining, DNA-PI staining and DNA ladder. Western blot was used to analyse mitochondrial and cytosolic cyto C, Bcl-X(L), caspase-3, actin protein and the level of tyrosine phosphorylation.</p><p><b>RESULTS</b>After exposure to STI571, K562 cells were induced to apoptosis. Tyrosine phosphorylation level of P210(BCR/ABL) and Bcl-X(L) was decreased. Caspase-3 was activated and there was an cytosolic accumulation of cyto C.</p><p><b>CONCLUSION</b>STI571 could rapidly decrease the tyrosine phosphorylation level of P210(BCR/ABL). The signal pathway mediated by the cytosolic translocation of mitochondrial cyto C was one of the mechanisms that STI571 inducing apoptosis. STI571 was an effective gene targeting therapeutic agent.</p>

Functions of cytochrome c in regulation of electron transfer and protein folding.

Cytochrome c, a "mobile electron carrier" of the mitochondrial respiratory chain, also occurs in detectable amounts in the cytosol, and can receive electrons from cytochromes present in endoplasmic reticulum and plasma membranes as well as from superoxide and ascorbate. The pigment was found to dissociate from mitochondrial membranes in liver and kidney when rats were subjected to heat exposure and starvation, respectively. Treating cytochrome c with hydroxylamine gives a partially deaminated product with altered redox properties; decreased stimulation of respiration by deficient mitochondria, increased reduction by superoxide, and complete loss of reducibility by plasma membranes. Mitochondria isolated from brown adipose tissue of cold-exposed rats are found to be sub-saturated with cytochrome c. The ability of cytochrome c to reactivate reduced ribonuclease is now reinterpreted as a molecular chaperone role for the hemoprotein.

Effect of cisplatin, rIFN-Y, LPS and MDP on release of H2O2, O2- and lysozyme from human monocytes in vitro.

Increased secretion of H2O2, O2- and lysozyme by human monocytes in vitro on treatment with cisplatin, rIFN-Y (interferon-Y), LPS (lipopolysaccharide) and MDP (muramyl dipeptide) is reported. It is suggested that increased production of these secretory products represent the activated state of monocytes. These in vitro activated monocytes could either kill the tumor cells via increased contact mediated cytolysis or cytolysis mediated via the release of the secretory products like H2O2, O2- and lysozyme.

Hemolysin production by Leptospira interrogans serovar canicola in a protein-free medium with hemin.

Leptospira interrogans serovar canicola strain moulton was grown to a high cell density in a protein-free medium. When hemin was added to this medium, hemolysin was produced. Hemolysin was not detected when other porphyrins or cytochrome C were substituted for hemin in the medium.