971 resultados para LiGaO2 substrates
Resumo:
Cell division, which leads to the birth of two daughter cells, is essential for the growth and development of all organisms. The reproduction occurs in a series of events separated in time, designated as the cell cycle. The cell cycle progression is controlled by the activity of cyclin-dependent kinases (CDK). CDKs pair with cyclins to become catalytically active and phosphorylate a broad range of substrates required for cell cycle progression. In addition to cyclins, CDKs are regulated by inhibitory and activating phosphorylation events, binding to CDK-inhibitory proteins (CKI), and also by subcellular localization. The control of the CDK activity is crucial in preventing unscheduled progression of the cell cycle with mistakes having potentially hazardous consequences, such as uncontrolled proliferation of the cells, a hallmark of cancer. The mammalian cell cycle is a target of several DNA tumor viruses that can deregulate the host s cell cycle with their viral oncoproteins. A human herpesvirus called Kaposi s sarcoma herpesvirus (KSHV) is implicated in the cause of Kaposi s sarcoma (KS) and lymphoproliferative diseases such as primary effusion lymphomas (PEL). KSHV has pirated several cell cycle regulatory genes that it uses to manipulate its host cell and to induce proliferation. Among these gene products is a cellular cyclin D homologue, called viral cyclin (v-cyclin) that can activate cellular CDKs leading to the phosphorylation of multiple target proteins. Intriguingly, PELs that are naturally infected with KSHV consistently express high levels of CDK inhibitor protein p27Kip1 and still proliferate actively. The aim of this study was to investigate v-cyclin complexes and their activity in PELs, and search for an explanation why CKIs, such as p27Kip1 and p21Cip1 are unable to inhibit cell proliferation in this type of lymphoma. In this study, we found that v-cyclin binds to p27Kip1 in PELs, and confirmed this novel interaction also in the overexpression models. We observed that p27Kip1 associated with v-cyclin was also phosphorylated by a v-cyclin-associated kinase and identified cellular CDK6 as the major kinase partner of v-cyclin responsible for this phosphorylation. Analysis of the p27Kip1 residues targeted by v-cyclin-CDK6 revealed that serine 10 (S10) is the major phosphorylation site during the latent phase of the KSHV replication cycle. This phosphorylation led to the relocalization of p27Kip1 to the cytoplasm, where it is unable to inhibit nuclear cyclin-CDK complexes. In the lytic phase of the viral replication cycle, the preferred phosphorylation site on p27Kip1 by v-cyclin-CDK6 changed to threonine 187 (T187). T187 phosphorylation has been shown to lead to ubiquitin-mediated degradation of p27Kip1 and downregulation of p27Kip1 was also observed here. v-cyclin was detected also in complex with p21Cip1, both in overexpression models and in PELs. Phosphorylation of p21Cip1 on serine 130 (S130) site by v-cyclin-CDK6 functionally inactivated p21Cip1 and led to the circumvention of G1 arrest induced by p21Cip1. Moreover, p21Cip1 phosphorylated by v-cyclin-associated kinase showed reduced binding to CDK2, which provides a plausible explanation why p21Cip1 is unable to inhibit cell cycle progression upon v-cyclin expression. Our findings clarify the mechanisms on how v-cyclin evades the inhibition of cell cycle inhibitors and suggests an explanation to the uncontrolled proliferation of KSHV-infected cells.
Resumo:
For the first time, we have fabricated and tested conductometric sensors based on oxidized liquid galinstan towards NO2 and NH3 gases at low operating temperatures. Galinstan based films on silicon substrates have been studied with two different loadings. Surface morphology of the films was investigated by means of field emission scanning electron microscopy (FESEM). The sensor with higher galinstan loading showed a better sensitivity, which can be attributed to a higher surface area, as confirmed by SEM. At 100°C, a detection limit as low as 1 and 20 ppm was measured for NO2 and NH3, respectively.
Resumo:
Generation of H2O2 by rat liver mitochondria with choline, glycerol 1-phosphate and proline as substrates has been shown by using high-concentration phosphate buffer. Rates obtained under these conditions were higher and more consistent as compared with the earlier reports with high-concentration mannitol/sucrose/Tris buffer. Sulphate ions could replace phosphate indicating a requirement for a high concentration of oxygen-containing anions. H2O2 generation was dependent on the presence of native mitochondria and substrate. Maximal rates with various substrates were found to be the same as with succinate. Values of Km and Vmax for H2O2 generation were considerably less than those obtained for respective dehydrogenase activities, measured by dye reduction. Scavengers of O2-. and OH. inhibited generation of H2O2. ATP, ADP, thyronine derivatives and a number of phenolic compounds also showed very potent inhibitory effects of H2O2 generation, whereas phenyl compound had no effect. Phenolic compounds did not have any effect on mitochondrial superoxide dismutase and choline dehydrogenase activities as well as on O2-. generation by the xanthine-xanthine oxidase system. Inhibition by phenolic compounds may have potential for regulation of the intracellular concentration of H2O2, that is not considered to have a "second messenger' function.
Resumo:
Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.
Resumo:
Secondary crops provide a means of assimilating some effluent nitrogen from eutrophic shrimp farm settlement ponds. However, a more important role may be their stimulation of beneficial bacterial nitrogen removal processes. In this study, bacterial biomass, growth and nitrogen removal capacity were quantified in shrimp farm effluent treatment systems containing vertical artificial substrates and either the banana shrimp Penaeus merguiensis (de Man) or the grey mullet, Mugil cephalus L. Banana shrimp were found to actively graze biofilm on the artificial substrates and significantly reduced bacterial biomass relative to a control (24.5 ± 5.6mgCm−2 and 39.2 ± 8.7mgCm−2, respectively). Bacterial volumetric growth rates, however, were significantly increased in the presence of the shrimp relative to the control 45.2±11.3mgCm−2 per day and 22.0±4.3mgCm−2 per day, respectively). Specific growth rate, or growth rate per cell, of bacteria was therefore appreciably stimulated by the banana shrimp. Nitrate assimilation was found to be significantly higher on grazed substrate biofilm relative to the control (223±54 mgNm−2 per day and 126±36 mg Nm−2 per day, respectively), suggesting that increased bacterial growth rate does relate to enhanced nitrogen uptake. Regulated banana shrimp feeding activity therefore can increase the rate of newbacterial biomass production and also the capacity for bacterial effluent nitrogen assimilation. Mullet had a negligible influence on the biofilm associated with the artificial substrate but reduced sediment bacterial biomass (224 ± 92 mgCm−2) relative to undisturbed sediment (650 ± 254 mgCm−2). Net, or volumetric bacterial growth in the sediment was similar in treatments with and without mullet, suggesting that the growth rate per cell of bacteria in grazed sediments was enhanced. Similar rates of dissolved nitrogen mineralisation werefound in sediments with and without mullet but nitrificationwas reduced. Presence of mullet increased water column suspended solids concentrations, water column bacterial growth and dissolved nutrient uptake. This study has shown that secondary crops, particularly banana shrimp, can play a stimulatory role in the bacterial processing of effluent nitrogen in eutrophic shrimp effluent treatment systems.
Resumo:
The aim of this project was to quantify differences between treated and untreated coir (coconut industrial residues) products and to identify differences in growth, yield and quality of cut flowers grown in different coir products. This has been brought about largely by the concern that some coir products, washed in low quality (saline) water may have detrimental effects on plant productivity and quality. There is concern in the flower production industry and among media suppliers, that lower quality products are favoured due to price alone, which as this project shows is a false economy. Specifically the project examined: • Differences in physical and chemical properties of treated and untreated coir along with another commonly used growing media in the flower industy; • Potential improvements in yield and quality of Gerbera (Gerbera jamesonii); • Potential differences in vase life of Gerbera as a result of the different growing media; and • Cost-benefit implications of treated (more expensive) coir substrate products versus untreated (less expensive) coir including any subsequent differences in yield and quality. By first examining the physical and some chemical properties of different coir substrates and other industry standard media, the researchers have been able to validate the concerns raised about the potential quality issues in coir based growing media. There was a great deal of variation in both the electrical conductivity and sodium contents. Physical properties were also variable as expected since manufacturers are able to target the specific physical preferences of plants through manipulation of the particle size distribution. A field trial was conducted under protected cropping practices in which three growing media were compared in terms of total productivity and also flower quality parameters such as stem length, flower diameter and vase life. The trial was a completely randomised design with the three growing media comprising treated coir discs, untreated coir discs and a pine bark coir mix. Four cultivars of Gerbera were assessed: Balance®; Carambole®; Dune® and Picobello®, all new products from Florist de Kwakel B.V., Denmark. Initial expansion from tissue culture was conducted at the Highsun Express Facility, Ormiston, Queensland. The trial included 12 replications of each cultivar in each media (a total of 144 plants) to ensure all data collected, and the derived conclusions were statistically rigorous. The coir supplied with no pre-treatment or buffering produced significantly less flowers than those grown in a pine bark coir mix or the pre-treated coir. Interestingly, the pine bark coir mix produced a greater number of flowers. However, the flowers produced in the pine bark coir mix were generally a shorter length stem. Productivity data, combined with flower quality data and component costs were all analysed through a cost/benefit economic model which showed that the greater revenue from better stem length outweighed the stem numbers, giving a cost benefit ratio of 2.58 for treated coir, 2.49 for untreated coir and 2.52 for pine bark coir mix. While this does not seem a large difference, when considering the number of plants a producer maintains can be upwards of 50,000 the difference in revenue would be, at a minimum $60,000 in this example. In conclusion, this project has found that there are significant effects on plant health, growth, yield and quality between those grown in treated and untreated coir. The outcome being growers can confidently invest in more expensive treated products with the assurance that benefits will outweigh initial cost. It is false economy to favour untreated coir products based on price alone. Producers should ensure they fully understand the production processes when purchasing growing media. Rather than targeting lower priced materials, it is recommended that quality be the highest priority in making this management decision. In making recommendations for future research and development it was important to consider conclusions from other researchers as well as those of the current project. It has been suggested that the media has greater longevity, which although not captured in this study could also lead to further cost efficiencies. Assessment of the products over a longer time period, and using a wider range of plant species are the major recommendations for further research to ensure greater understanding as to the importance in choosing the right growing media to meet specific needs.
Heat exposure and hypothyroid conditions decrease hydrogen peroxide generation in liver mitochondria
Resumo:
Exposure of rats to heat (39 +/- 1 degree C) decreased H2O2 generation in mitochondria of the liver, but not of the kidney or the heart. The effect was obtained with three substrates, succinate, glycerol 1-phosphate and choline, with a decrease to 50% in the first 2-3 days of exposure, and a further decrease on longer exposure. The dehydrogenase activity with only glycerol 1-phosphate decreased, which is indicative of the hypothyroid condition, whereas choline dehydrogenase activity remained unchanged and that of succinate dehydrogenase decreased on long exposure. The serum concentration of thyroxine decreased in heat-exposed rats. Thyroxine treatment of rats increased H2O2 generation. Hypothyroid conditions obtained by treatment with propylthiouracil or thyroidectomy caused a decrease in H2O2 generation and changes in dehydrogenase activities similar to those with heat exposure. Treatment of heat-exposed or thyroidectomized rats with thyroxine stimulated H2O2 generation by a mechanism apparently involving fresh protein synthesis. The results indicate that H2O2 generation in mitochondria of heat-exposed animals is determined by thyroid status.
Resumo:
Growing crystals with selected structure and preferred orientations oil seed substrates is crucial for a wide variety of applications. Although epitaxial or textured film growth of a polymorph whose structure resembles the seed crystal structure is well-known, growing oriented nanocrystal arrays or more than one polymorph, selectable one at a time, from the same seed has not been realized. Here, we demonstrate for the first time the exclusive growth of oriented nanocrystal arrays of two titania polymorphs from a titanate crystal by chemically activating respective polymorph-mimicking crystallographic facets in the seed. The oriented titania nanocrystal arrays exhibit significantly higher photocatalytic activity than randomly oriented polymorphs. Our approach of chemically sculpting oriented nanocrystal polymorph arrays could be adapted to other materials systems to obtain novel properties.
Resumo:
Background: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. Results: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. Conclusion: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.
Resumo:
Linalool-8-monoxygenase, a typical bacterial P-450 heme thiolase, shows a high degree of substrate specificity towards linalool. The active site of the pure enzyme has been probed with a large number of substrate analogues with systematic alterations or conformational variations in the linalool molecule. The comparison of three parameters, the mo→mos conversion of the enzyme as a result of substrate binding monitored at 392 nm, theK D of the analogues giving information about energies of association and the relative turnover as substrate have given information about the space-filling characteristics of the substrates in the enzyme cleft, the number of contacts the molecules make with the respective domains of the enzyme and the distance of the site undergoing hydroxylation from the oxygen site, respectively. The data permit the conclusion that linalool makes contact with the enzyme by hydrogen bonding with the hydroxyl group as well through hydrophobic association with all the eight carbons carrying hydrogen in the molecules.
Resumo:
Surface-enhanced Raman scattering (SERS) of pyridine adsorbed on ultrathin nanocrystalline Au and Ag films generated at the liquid-liquid interface has been investigated. The shifts and intensification of bands formed with these films comprising metal nanoparticles are comparable to those found with other types of Au and Ag substrates. SERS of rhodamine 6G adsorbed on Ag films has also been studied. The results demonstrate that nanocrystalline metal films prepared by the simple method involving the organic-aqueous interface can be used effectively for SERS investigations.
Resumo:
Gene therapy is a promising novel approach for treating cancers resistant to or escaping currently available modalities. Treatment approaches are based on taking advantage of molecular differences between normal and tumor cells. Various strategies are currently in clinical development with adenoviruses as the most popular vehicle. Recent developments include improving targeting strategies for gene delivery to tumor cells with tumor specific promoters or infectivity enhancement. A rapidly developing field is as well replication competent agents, which allow improved tumor penetration and local amplification of the anti-tumor effect. Adenoviral cancer gene therapy approaches lack cross-resistance with other treatment options and therefore synergistic effects are possible. This study focused on development of adenoviral vectors suitable for treatment of various gynecologic cancer types, describing the development of the field from non-replicating adenoviral vectors to multiple-modified conditional replicating viruses. Transcriptional targeting of gynecologic cancer cells by the use of the promoter of vascular endothelial growth factor receptor type 1 (flt-1) was evaluated. Flt-1 is not expressed in the liver and thus an ideal promoter for transcriptional targeting of adenoviruses. Our studies implied that the flt-1 promoter is active in teratocarcinomas.and therefore a good candidate for development of oncolytic adenoviruses for treatment of this often problematic disease with then poor outcome. A tropism modified conditionally replicating adenovirus (CRAd), Ad5-Δ24RGD, was studied in gynecologic cancers. Ad5-Δ24RGD is an adenovirus selectively replication competent in cells defective in the p16/Rb pathway, including many or most tumor cells. The fiber of Ad5-Δ24RGD contains an integrin binding arginine-glycine-aspartic acid motif (RGD-4C), allowing coxackie-adenovirus receptor independent infection of cancer cells. This approach is attractive because expression levels of CAR are highly variable and often low on primary gynecological cancer cells. Oncolysis could be shown for a wide variety of ovarian and cervical cancer cell lines as well as primary ovarian cancer cell spheroids, a novel system developed for in vitro analysis of CRAds on primary tumor substrates. Biodistribution was evaluated and preclinical safety data was obtained by demonstrating lack of replication in human peripheral blood mononuclear cells. The efficicacy of Ad5-Δ24RGD was shown in different orthotopic murine models including a highly aggressive intraperitoneal model of disseminated ovarian cancer cells, where Ad5-Δ24RGD resulted in complete eradication of intraperitoneal disease in half of the mice. To further improve the selectivity and specificity of CRAds, triple-targeted oncolytic adenoviruses were cloned, featuring the cyclo-oxygenase-2 (cox-2) promoter, E1A transcomplementation and serotype chimerism. Those viruses were evaluated on ovarian cancer cells for specificity and oncolytic potency with regard to two different cox2 versions and three different variants of E1A (wild type, delta24 and delta2delta24). Ad5/3cox2Ld24 emerged as the best combination due to enhanced selectivity without potency lost in vitro or in an aggressive intraperitoneal orthotopic ovarian tumor model. In summary, the preclinical therapeutic efficacy of the CRAds tested in this study, taken together with promising biodistribution and safety data, suggest that these CRAds are interesting candidates for translation into clinical trials for gynecologic cancer.
Resumo:
Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.
Functional changes in rat liver mitochondria on administration of 2-methyl-4-dimethylaminoazobenzene
Resumo:
Administration of 2-methyl-4-dimethylaminobenzene in the diet (0.1%, w/w) for 85-90 days doubled the content of mitochondria in the livers of rats. The azodye was covalently bound to liver proteins, and about 15% of the amount found in liver was associated with the mitochondrial fraction. Mitochondria isolated from the livers of azodye-fed animals showed drastically lowered ability to oxidize NAD+-linked substrates. The inhibited electron-transfer step was the reduction of ubiquinone. The organelles showed a large increase in succinate oxidase activity. The activity of cytochrome oxidase and the content of cytochrome aa3 were substantially higher in these organelles. Azodye-fed animals showed depressed serum cholesterol concentrations. The content of ubiquinone in liver also registered a small increase.
Resumo:
The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.