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Mercer Kelly E.; Ahn Christina E.; Coke Amanda; Compadre Cesar M.; Drake Richard R. 《Protein engineering, design & selection : PEDS》2002,15(11):903-911
Understanding the functional and mechanistic properties of themulti-substrate herpes simplex virus type-1 thymidine kinase(HSV-1 TK) remains critical to defining its role as a majorpharmacological target in herpesvirus and gene therapies forcancer. An inherent limitation of the activity of HSV-TK isthe >70-fold difference in the Kms for phosphorylation ofthymidine over the pro-drug ganciclovir (GCV). To engineer anHSV-1 TK isoform that is specific for GCV as the preferred substrate,16 site-specific mutants were generated. The mutations wereconcentrated at conserved residues involved in nucleoside basebinding, Gln125 and near sites 3 and 4 involved in catalysisand substrate binding. The substrate preferences of each mutantenzyme were compared with wild-type HSV-1 TK. One mutant, termedQ7530 TK, had a lower Km for GCV than thymidine. Expressionof the Q7530 TK in tumor cells indicated comparable metabolismto and improved sensitivity to GCV over wild-type HSV-1 TK,with minimal thymidine phosphorylation activity. A molecularmodeling simulation of the different HSV-1 TK active-sites wasdone for GCV and thymidine binding. It was concluded that mutationsat Gln125 and near site 4, especially at Ala168, were responsiblefor loss of deoxypyrimidine substrate binding. 相似文献
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Dorai C. Wang G. Jain A.K. Mercer C. 《IEEE transactions on pattern analysis and machine intelligence》1998,20(1):83-89
Automatic 3D object model construction is important in applications ranging from manufacturing to entertainment, since CAD models of existing objects may be either unavailable or unusable. We describe a prototype system for automatically registering and integrating multiple views of objects from range data. The results can then be used to construct geometric models of the objects. New techniques for handling key problems such as robust estimation of transformations relating multiple views and seamless integration of registered data to form an unbroken surface have been proposed and implemented in the system. Experimental results on real surface data acquired using a digital interferometric sensor as well as a laser range scanner demonstrate the good performance of our system 相似文献
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LP Mercer DS Kelley HM Bundrant AU Haq LL Humphries 《Canadian Metallurgical Quarterly》1996,126(12):3128-3135
The histaminergic system (histamine and its H1-receptor) of the central nervous system has been implicated in control of food intake. The reported studies were designed to examine the effects of food restriction and very low (1%) protein diets on central nervous system H1-receptors in male and female rats. In a series of experiments, groups of rats were freely fed a 25% protein diet, a 1% protein diet, or fed the 25% protein diet at 4 g/100 g body weight for 14-20 d. When freely fed 25% protein diets, females had higher whole-brain H1-receptor binding than males on d 1 (female 122.36 +/- 4.53 and male 65.78 +/- 3.82 pmol/g protein; P < 0.001). Changing diets affected central H1-receptor binding in both males and females (P < 0.003). When rats were fed both restricted levels of food and 1% protein diets, the receptor binding of males increased by d 5 whereas that of females decreased by d 5 (P < 0.001). When fed 1% protein diets, females had decreased H1-receptor binding (98.4 +/- 2.38 pmol/g protein) and that in males increased to 119.81 +/- 5.09 pmol/g protein. After 15 d, females had eaten significantly more food than males: females 166 +/- 4.9 g, males 124 +/- 1.9 g (P< 0.0007). Males had a significantly greater weight loss than females: males -28.8 +/- 2.6 g, females -17.08 +/- 0.97 g (P < 0.0007). When fed restricted diets, females had decreased H1-receptor binding (93.81 +/- 5.58 pmol/g) whereas binding in males increased to 111.27 +/- 8.55 pmol/g. Preliminary saturation binding studies indicated that restricted food intake lowered receptor density (females consuming 25% protein: 715 +/- 30 pmol/g protein; female restricted: 467 +/- 28 pmol/g protein, P < 0.05), while 1% protein increased receptor sensitivity, i.e., lowered KD (males consuming 25% protein: 15.3 +/- 1.8 nmol; males fed low protein: 2.8 +/- 0.27 nmol). This study suggests that dietary manipulation affects central H1-receptor binding in a gender-specific manner, thereby modulating central histaminergic activity during food or protein deficit. 相似文献
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P Factor F Saldias K Ridge V Dumasius J Zabner HA Jaffe G Blanco M Barnard R Mercer R Perrin JI Sznajder 《Canadian Metallurgical Quarterly》1998,102(7):1421-1430
The gastrointestinal hormone, glucagon-like peptide-1(7-36)amide (GLP-1) is released after a meal. The potency of synthetic GLP-1 in stimulating insulin secretion and in inhibiting glucagon secretion indicates the putative physiological function of GLP-1. In vitro, the nonmammalian peptide, exendin(9-39)amide [ex(9-39)NH2], is a specific and competitive antagonist of GLP-1. This in vivo study examined the efficacy of ex(9-39)NH2 as an antagonist of exogenous GLP-1 and the physiological role of endogenous GLP-1. Six healthy volunteers underwent 10 experiments in random order. In each experiment, a 30-min period of euglycemia was followed by an intravenous infusion of glucose for 150 min that established a stable hyperglycemia of 8 mmol/liter. There was a concomitant intravenous infusion of one of the following: (1) saline, (2) GLP-1 (for 60 min at 0.3 pmol . kg-1 . min-1 that established physiological postprandial plasma levels, and for another 60 min at 0.9 pmol . kg-1 . min-1 to induce supraphysiological plasma levels), (3-5) ex(9-39)NH2 at 30, 60, or 300 pmol . kg-1 . min-1 + GLP-1, (6-8) ex(9-39)NH2 at 30, 60, or 300 pmol . kg-1 . min-1 + saline, (9 and 10) GIP (glucose-dependent insulinotropic peptide; for 60 min at 0.8 pmol . kg-1 . min-1, with saline or ex(9-39)NH2 at 300 pmol . kg-1 . min-1). Each volunteer received each of these concomitant infusions on separate days. ex(9-39)NH2 dose-dependently reduced the insulinotropic action of GLP-1 with the inhibitory effect declining with increasing doses of GLP-1. ex(9-39)NH2 at 300 pmol . kg-1 . min-1 blocked the insulinotropic effect of physiological doses of GLP-1 and completely antagonized the glucagonostatic effect at both doses of GLP-1. Given alone, this load of ex(9-39)NH2 increased plasma glucagon levels during euglycemia and hyperglycemia. It had no effect on plasma levels of insulin during euglycemia but decreased plasma insulin during hyperglycemia. ex(9-39)NH2 did not alter GIP-stimulated insulin secretion. These data indicate that in humans, ex(9-39)NH2 is a potent GLP-1 antagonist without any agonistic properties. The pancreatic A cell is under a tonic inhibitory control of GLP-1. At hyperglycemia, the B cell is under a tonic stimulatory control of GLP-1. 相似文献
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PJ Morgan CA Webster JG Mercer AW Ross DG Hazlerigg A MacLean P Barrett 《Canadian Metallurgical Quarterly》1996,137(9):4018-4026
The purpose of this study was to determine whether the cells of the ovine pars tuberalis (PT) secrete a factor(s) that can influence the activity of cells in the pars distalis (PD). By Northern blotting of total RNA isolated from PD cells that had been stimulated in the presence of cycloheximide (10 micrograms/ml), PT cell-conditioned medium was shown to induce a significant increase in the expression of the early response gene, c-fos, above both PD cell-conditioned and nonconditioned medium control levels (P < 0.05). Although forskolin (5 microM) induced a weak increase in c-fos expression in PD cells, the effect of PT medium conditioned in the presence of forskolin enhanced this expression more than additively (P < 0.05); furthermore, this effect was reversed by melatonin. These results are consistent with the release of a factor(s) from the PT, which for simplicity we have called tuberalin. This factor was released from PT cells in a time-dependent and cycloheximide-sensitive manner and was resistant to heating at 100 C for 10 min. Tuberalin activity could be size-fractionated using molecular size cut-off filters to produce activity in both the 1- to 10-kDa and more than 10-kDa size ranges. The activities in both of these fractions were sensitive to trypsin degradation and, therefore, appeared to be peptidergic. However, it was not clarified whether the biological activities were due to one or two components. Tuberalin also induced c-fos expression in other cell types, including GH3 and NIH3T3 cells. Dual labelling of PD cells by in situ hybridization using riboprobes for c-fos and PRL demonstrated that both the less than and more than 10-kDa fractions of tuberalin activated c-fos expression in some, but not all, lactotrophs in PD cell cultures, suggesting that a primary function of the PT is to regulate the activity of lactotrophs. This was supported further by enhanced secretion of PRL from PD cells in the presence of either PT-conditioned medium or PT cells in coculture. In addition, PT-conditioned medium was found to increase c-fos in a second cell type, which did not hybridize positively for PRL, indicating the existence of other endocrine interactions between the PT and PD. 相似文献