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81.
Shrivastava A Tiwari M Sinha RA Kumar A Balapure AK Bajpai VK Sharma R Mitra K Tandon A Godbole MM 《The Journal of biological chemistry》2006,281(28):19762-19771
Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-L-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway. 相似文献
82.
Shinde P Dass RS Garg AK Chaturvedi VK Kumar R 《Biological trace element research》2006,112(3):247-262
Forty weaned male guinea pigs of 208.20±6.62 g mean body weight were divided into 4 groups of 10 animals in a randomized block
design. All of the guinea pigs were fed a basal diet [25% ground maize hay, 30% ground maize grain, 22% ground chickpea (Cicer arietinum L.), 9.5% deoiled rice bran, 6% soybean meal, 6% fish meal, 1.45% mineral supplement (without Zn) and 0.05% ascorbic acid]
and available green fodder. Group I served as the control (no Zn supplementation), whereas 20 ppm Zn was added in the diet
in groups II, III, and IV either as zinc sulfate (ZnSO4), zinc amino acid complex (ZAAC), and ZnSO4
+ ZAAC in equal parts, respectively. Experimental feeding lasted for 70 d, including a 3-d digestibility trial. Blood was collected
through cardiac puncture from four animals in each group at d 0 and subsequently at the end of experimental feeding. After
40 d of experimental feeding, four animals from each group were injected with 0.4 mL of Brucella abortus cotton strain-19 vaccine to assess the humoral immune response of the animals. After 10 wk of study, four animals from each
group were sacrificed to study the concentration of Zn, Cu, Co, Fe, and Mn in the liver, pancreas and spleen. Results revealed
no significant difference in the feed intake, body weight gain, and digestibility of the nutrients, except for crude protein
(CP) digestibility, which was significantly (p<0.05) lower in group IV. Although concentrations of serum glucose, Ca, and P and the albumin:globulin (A:G) ratio were similar
in the different groups, the total protein, albumin, and serum alkaline phosphatase activity were higher in all of the Zn-supplemented
groups on d 70. The serum Zn levels at the end of experimental feeding were significantly higher in groups II and III, whereas
serum Mn levels were found to be significantly (p<0.05) higher in groups III and IV. The organ weights (as percentage of body weights) did not show any differences among the
treatment groups. Although the Mn concentration was significantly (p<0.05) higher in the pancreas, the Cu concentration was significantly (p<0.05) reduced in the spleen in all of the Zn-supplemented groups. The humoral immune response (antibody titer values) on
d 14 of vaccination was significantly (p<0.05) higher in all of the Zn-supplemented groups. It was concluded that the 20-ppm level of Zn in the diet might be adequate
for growth and nutrient utilization in guinea pigs, but supplementation of 20-ppm zinc significantly improved the immune response
and impact was more prominent with the ZAAC (organic source) compared to ZnSO4 (inorganic source). 相似文献
83.
It is well-known that secondary metabolite production is repressed by excess nitrogen substrate available in the fermentation media. Although the nitrogen catabolite repression has been known, quantitative process models have not been reported to represent this phenomenon in complex medium. In this paper, we present a cybernetic model for rifamycin B production via Amycolatopsis mediterranei S699 in complex medium, which is typically used in industry. Nitrogen substrate is assumed to be present in two forms in the medium; available nitrogen (S
ANS) such as free amino acids and unavailable nitrogen (S
UNS) such as peptides and proteins. The model assumes that an inducible enzyme catalyzes the conversion of S
UNS to S
ANS. Although S
ANS is required for growth and product formation, high concentrations were found to inhibit rifamycin production. To experimentally validate the model, five different organic nitrogen sources were used that differ in the ratio of S
ANS/S
UNS. The model successfully predicts higher rifamycin B productivity for nitrogen sources that contain lower initial S
ANS. The higher productivity is attributed to the sustained availability of S
ANS at low concentration via conversion of S
UNS to S
ANS, thereby minimizing the effects of nitrogen catabolite repression on rifamycin production. The model can have applications in model-based optimization of substrate feeding recipe and in monitoring and control of fed batch processes. 相似文献
84.
Body fragmentation, in some animal groups, is a mechanism for survival and asexual reproduction. Lumbriculus variegatus (Müller, 1774), an aquatic oligochaete worm, is capable of regenerating into complete individuals from small body fragments
following injury and reproduces primarily by asexual reproduction. Few studies have determined the cellular mechanisms that
underlie fragmentation, either regenerative or asexual. We utilized boric acid treatment, which blocks regeneration of segments
in amputated fragments and blocks architomic fission during asexual reproduction, to investigate mechanistic relationships
and differences between these two modes of development. Neural morphallaxis, involving changes in sensory fields and giant
fiber conduction, was detected in amputated fragments in the absence of segmental regeneration. Furthermore, neural morphallactic
changes occurred as a result of developmental mechanisms of asexual reproduction, even when architomy was prevented. These
results show that fragmentation in L. variegatus, during injury or asexual reproduction, employs developmental and morphallactic processes that can be mechanistically dissociated
by boric acid exposure. In regeneration following injury, compensatory morphallaxis occurred in response to fragmentation.
In contrast, anticipatory morphallaxis was induced in preparation for fragmentation during asexual reproduction. Thus, various
forms of regeneration in this lumbriculid worm can be activated independently and in different developmental contexts. 相似文献
85.
Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells 总被引:4,自引:0,他引:4
Katkov II Kim MS Bajpai R Altman YS Mercola M Loring JF Terskikh AV Snyder EY Levine F 《Cryobiology》2006,53(2):194-205
We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation. 相似文献
86.
Prashant M Bapat Debasish Das Sujata V Sohoni Pramod P Wangikar 《Microbial cell factories》2006,5(1):32-14
Background
Industrial fermentation typically uses complex nitrogen substrates which consist of mixture of amino acids. The uptake of amino acids is known to be mediated by several amino acid transporters with certain preferences. However, models to predict this preferential uptake are not available. We present the stoichiometry for the utilization of amino acids as a sole carbon and nitrogen substrate or along with glucose as an additional carbon source. In the former case, the excess nitrogen provided by the amino acids is excreted by the organism in the form of ammonia. We have developed a cybernetic model to predict the sequence and kinetics of uptake of amino acids. The model is based on the assumption that the growth on a specific substrate is dependent on key enzyme(s) responsible for the uptake and assimilation of the substrates. These enzymes may be regulated by mechanisms of nitrogen catabolite repression. The model hypothesizes that the organism is an optimal strategist and invests resources for the uptake of a substrate that are proportional to the returns. 相似文献87.
88.
Bansal S Srivastava A Mukherjee G Pandey R Verma AK Mishra P Kundu B 《FASEB journal》2012,26(3):1161-1171
Thermophilic l-asparaginases display high stability and activity at elevated temperatures. However, they are of limited use in leukemia therapy because of their low substrate affinity and reduced activity under physiological conditions. In an attempt to combine stability with activity at physiological conditions, 3 active-site mutants of Pyrococcus furiosus l-asparaginase (PfA) were developed. The mutants, specifically K274E, showed improved enzymatic properties at physiological conditions as compared to the wild type. All variants were thermodynamically stable and resistant to proteolytic digestion. None of the enzymes displayed glutaminase activity, a highly desirable therapeutic property. All variants showed higher and significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli l-asparaginase. Our study revealed that increased substrate accessibility through the active site loop plays a major role in determining activity. A new mechanistic insight has been proposed based on molecular dynamics simulated structures, where dynamic flipping of a critical Tyr residue is responsible for the activity of thermophilic l-asparaginases. Our study not only resulted in development of PfA mutants with combination of desirable properties but also gave a mechanistic insight about their activity. 相似文献
89.
Previously, we have demonstrated that the chloride channel ClC-2 modulates intestinal mucosal barrier function. In the present study, we investigated the role of ClC-2 in epithelial barrier development and maintenance in Caco-2 cells. During early monolayer formation, silencing of ClC-2 with small interfering (si)RNA led to a significant delay in the development of transepithelial resistance (TER) and disruption of occludin localization. Proteomic analysis employing liquid chromatography-mass spectrometry /mass spectrometry revealed association of ClC-2 with key proteins involved in intracellular trafficking, including caveolin-1 and Rab5. In ClC-2 siRNA-treated cells, occludin colocalization with caveolin-1 was diffuse and in the subapical region. Subapically distributed occludin in ClC-2 siRNA-treated cells showed marked colocalization with Rab5. To study the link between ClC-2 and trafficking of occludin in confluent epithelial monolayers, a Caco-2 cell clone expressing ClC-2 short hairpin (sh)RNA was established. Disruption of caveolae with methyl-β-cyclodextrin (MβCD) caused a marked drop in TER and profound redistribution of caveolin-1-occludin coimmunofluorescence in ClC-2 shRNA cells. In ClC-2 shRNA cells, focal aggregations of Rab5-occludin coimmunofluorescence were present within the cytoplasm. Wortmannin caused an acute fall in TER in ClC-2 shRNA cells and subapical, diffuse redistribution of Rab5-occludin coimmunofluorescence in ClC-2 shRNA cells. An endocytosis and recycling assay for occludin revealed higher basal rate of endocytosis of occludin in ClC-2 shRNA cells. Wortmannin significantly reduced the rate of recycling of occludin in ClC-2 shRNA cells. These data clearly indicate that ClC-2 plays an important role in the modulation of tight junctions by influencing caveolar trafficking of the tight junction protein occludin. 相似文献
90.
Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (-43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2-8°C for more than 3 months. IC(50) value of Ambisome (0.18 μg/mL) was comparatively similar to F-1a (0.17 μg/mL) and F-2a (0.16 μg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products. 相似文献