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231.
Bahrami G Mirzaeei S Kiani A Mohammadi B 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,823(2):213-217
A simple, fast, and sensitive high performance liquid chromatographic (HPLC) assay was developed for quantitation of lamivudine in human serum. Lamivudine is polar compound and its extraction from the human serum in previously published HPLC methods involved either protein precipitation or solid phase extraction techniques. However, existence of endogenous peaks which interfere with the drug or appeared as late eluting peaks and lead to long run time of analysis has been reported. Application of either an ion pairing agent in the mobile phase or time consuming column purge has been used in the published methods. Present paper describes liquid - liquid extraction of lamivudine and internal standard (famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M) containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of 2.2 mL/min. The analysis was performed on a column (150 mm x 6 mm i.d.) which was packed with 5 microm particles of ODS packing material. Under these conditions no interference in the assay from any endogenous substance was observed. The limit of quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were also studied and the technique was shown to be selective and linear into the concentration range of 5-2500 ng/mL. This method has been used in two randomized crossover bioequivalence studies of 100 and 150 mg lamivudine preparations in 12 and 24 healthy volunteers, respectively. 相似文献
232.
Bailey CM Khalkhali-Ellis Z Kondo S Margaryan NV Seftor RE Wheaton WW Amir S Pins MR Schutte BC Hendrix MJ 《The Journal of biological chemistry》2005,280(40):34210-34217
233.
Amira G Ifat M Tal A Hana B Shmuel G Rachel A 《Journal of experimental botany》2005,56(419):2443-2452
With the general aim of elevating the content of the essential amino acid methionine in vegetative tissues of plants, alfalfa (Medicago sativa L.) and tobacco plants, as well as BY2 tobacco suspension cells, were transformed with a beta-zein::3HA gene under the 35S promoter of cauliflower mosaic virus encoding a rumen-stable methionine-rich storage protein of 15 kDa zein. To examine whether soluble methionine content limited the accumulation of the 15 kDa zein::3HA, methionine was first added to the growth medium of the different transgenic plants and the level of the alien protein was determined. Results demonstrated that the added methionine enhanced the accumulation of the 15 kDa zein::3HA in transgenic alfalfa and tobacco BY2 cells, but not in whole transgenic tobacco plants. Next, the endogenous levels of methionine were elevated in the transgenic tobacco and alfalfa plants by crossing them with plants expressing the Arabidopsis cystathionine gamma-synthase (AtCGS) having significantly higher levels of soluble methionine in their leaves. Compared with plants expressing only the 15 kDa zein::3HA, transgenic alfalfa co-expressing both alien genes showed significantly enhanced levels of this protein concurrently with a reduction in the soluble methionine content, thus implying that soluble methionine was incorporated into the 15 kDa zein::3HA. Similar phenomena also occurred in tobacco, but were considerably less pronounced. The results demonstrate that the accumulation of the 15 kDa zein::3HA is regulated in a species-specific manner and that soluble methionine plays a major role in the accumulation of the 15 kDa zein in some plant species but less so in others. 相似文献
234.
Regulatory mechanisms for human CYP27A1 enzyme have not yet been fully investigated. Our approach was to add different hormones and cytokines to cultured human monocyte-derived macrophages, and assess the effects on the CYP27A1 by measuring the production of 27-hydroxylated cholesterol in the media. Of the different hormones and cytokines tested, only transforming growth factor beta1 (TGF-beta1) had a clear effect on CYP27A1. Further experiments showed a significant increase in 27-hydroxylated cholesterol products (27-hydroxycholesterol and 3beta-hydroxy-5-cholestenoic acid). A concomitant increase in CYP27A1 mRNA levels was also seen and this positive effect was confirmed using a human CYP27A1 luciferase reporter gene expressed in HepG2 cells. Experiments with progressive deletion/luciferase reporter gene constructs indicated that a TGF-beta1 responsive sequence might be localized in a region about 400 bp upstream of the CYP27A1 translation start. The possibility is discussed that induction of CYP27A1 by TGF-beta1 may be responsible for some of the anti-atherogenic properties of this cytokine. 相似文献
235.
A variety of biophysical methods used to study proteins requires protein modification using conjugated molecular probes. Cysteine is the main residue that can be modified without the risk of altering other residues in the protein chain. It is possible to label several cysteines in a protein using highly selective labeling reactions, if the cysteines react at very different rates. The reactivity of a cysteine residue introduced into an exposed surface site depends on the fraction of cysteine in the deprotonated state. Here, it is shown that cysteine reactivity differences can be effectively predicted by an electrostatic model that yields site-specifically the fractions of cysteinate. The model accounts for electrostatic interactions between the cysteinyl anion and side chains, the local protein backbone, and water. The energies of interaction with side chains and the main chain are calculated by using the two different dielectric constants, 40 and 22, respectively. Twenty-six mutants of Escherichia coli adenylate kinase were produced, each containing a single cysteine at the protein surface, and the rates of the reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) were measured. Cysteine residues were chosen on the basis of locations that were expected to allow modification of the protein with minimal risk of perturbing its structure. The reaction rates spanned a range of 6 orders of magnitude. The correlation between predicted fractions of cysteinate and measured reaction rates was strong (R = 92%) and especially high (R = 97%) for cysteines at the helix termini. The approach developed here allows reasonably fast, automated screening of protein surfaces to identify sites that permit efficient preparations of double- or triple-labeled protein. 相似文献
236.
Interference with heparin binding and oligomerization creates a novel anti-inflammatory strategy targeting the chemokine system 总被引:4,自引:0,他引:4
Johnson Z Kosco-Vilbois MH Herren S Cirillo R Muzio V Zaratin P Carbonatto M Mack M Smailbegovic A Rose M Lever R Page C Wells TN Proudfoot AE 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(9):5776-5785
A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents. 相似文献
237.
Yoram Shechter Marina Mironchik Shimon Amir Ben-Ami Sela Haim Tsubery Hailin Zheng Mati Fridkin 《International journal of peptide research and therapeutics》2006,12(2):121-129
Polymyxin B (PMXB) blocks the action of insulin on glucose uptake in vitro. In vivo, it reverses hypoglycemia induced by exogenous insulin. Here we have treated mature male rats daily with PMXB over a period of two weeks. This therapy has decreased body weight by 11%, adipose fat mass by 46% and triglyceride levels by 39%, with no indication of liver or kidney toxicity. Two suboptimal parameters, however, were a decrease in food intake in the first week of treatment and some increase in fasting glucose levels. We have screened for PMXB-analogs having less associating affinity with rat-muscle phospholipids, and revealed that the same therapy using PMXB-derived peptide (nona-PMXB) is most optimal. This PMXB-analog is devoid of antibacterial activity and is four times less toxic than PMXB. Nona-PMXB therapy lower by 10, 32, 35 and 6% body weight gain, fat mass, circulating triglycerides and fasting glucose levels, respectively, in spite of normal or even elevated food intake in nona-PMXB treated rats. In summary, we found that nona-PMXB therapy is capable if inducing leanness in mature rats, particularly at the expense of decreasing fat-mass in adipose tissue. By and large, we suggest that lowering the action of insulin, on fat build-up solely, may be a therapeutically feasible task to fight with human adiposity in the future. 相似文献
238.
The nicotinic acetylcholine receptor Dalpha7 is required for an escape behavior in Drosophila
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Acetylcholine is the major excitatory neurotransmitter in the central nervous system of insects. Mutant analysis of the Dalpha7 nicotinic acetylcholine receptor (nAChR) of Drosophila shows that it is required for the giant fiber-mediated escape behavior. The Dalpha7 protein is enriched in the dendrites of the giant fiber, and electrophysiological analysis of the giant fiber circuit showed that sensory input to the giant fiber is disrupted, as is transmission at an identified cholinergic synapse between the peripherally synapsing interneuron and the dorsal lateral muscle motor neuron. Moreover, we found that gfA1, a mutation identified in a screen for giant fiber defects more than twenty years ago, is an allele of Dalpha7. Therefore, a combination of behavioral, electrophysiological, anatomical, and genetic data indicate an essential role for the Dalpha7 nAChR in giant fiber-mediated escape in Drosophila. 相似文献
239.
An investigation was conducted to evaluate the ability of Deferasirox (ICL670 or Exjade) following the distribution of cadmium
salt in male Wistar rats. Cadmium was introduced to several groups of weanling male Wistar rats through different means, by
act of drinking, feeding. A control group was fed on a diet containing normal level of iron. After a period of 30 days, all
the rats administered cadmium were severely anemic and showed toxicity symptoms through loss of hair and increasing in cadmium
and reduction in iron levels in blood. Chelation therapy was carried out to remove the toxic element from the body. The ability
of Deferasirox chelator in removing cadmium was investigated this chelator for 1 week to the remaining rats of similar groups.
The results showed that the cadmium level present in blood was significantly reduced and at the same time, iron concentration
returned to the normal level. It was concluded that Deferasirox chelator is able to remove cadmium from the body and could
be used for the treatment of complications and eradication of symptoms of cadmium intoxication. 相似文献
240.
The seaweed Ulva lactuca L. was spray cultured by mariculture effluents in a mattress‐like layer, held in air on slanted boards by plastic netting. Air‐agitated seaweed suspension tanks were the reference. Growth rate, yield, and ammonia‐N removal rate were 11.8% · d?1, 171 g fresh weight (fwt) · m?2 · d?1, and 5 g N · m?2 · d?1, respectively, by the spray‐cultured U. lactuca, and 16.9% · d?1, 283 g fwt · m?2 · d?1, and 7 g N · m?2 · d?1, respectively, by the tank U. lactuca. Biomass protein content was similar in both treatments. Dissolved oxygen in the fishpond effluent water was raised by >3 mg · L?1 and pH by up to half a unit, upon passage through both culture systems. The data suggest that spray‐irrigation culture of U. lactuca in this simple green‐mattress‐like system supplies the seaweed all it needs to grow and biofilter at rates close to those in standard air‐agitated tank culture. 相似文献