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1.
To characterize 25-hydroxyvitamin D3 24-hydroxylase and 25-hydroxyvitamin D3 1-hydroxylase, the activities of the two enzymes were measured in the presence of two types of inhibitors. The effect of protein synthesis inhibitors on 25-hydroxyvitamin D3-stimulated 24-hydroxylase activity in 1-hydroxylating rat kidneys perfused in vitro was tested. Actinomycin D (4 microM) and cycloheximide (10 microM) each abolished 25-hydroxyvitamin D3 24-hydroxylase synthesis when added at the start of perfusion but not when added 4 h later; they did not affect 25-hydroxyvitamin D3 1-hydroxylase activity. The effects of cytochrome P-450 inhibitors on the two enzyme activities were then studied in vivo. Metyrapone and SKF-525A (50 mg/kg body weight) each inhibited 25-hydroxyvitamin D3 24-hydroxylase at 6 and 24 h; in contrast 1-hydroxylase increased and was 5 times the control value at 24 h. Finally, the in vitro effects of six cytochrome P-450 inhibitors at concentrations ranging from 10(-7) to 10(-3) M on enzyme activities in renal mitochondrial preparations were compared. Both enzymes were inhibited by all of the inhibitors, but inhibition of 25-hydroxyvitamin D3 24-hydroxylase was consistently greater than that of 25-hydroxyvitamin D3 1-hydroxylase. These studies demonstrate that 24-hydroxylation and 1-hydroxylation respond differently to protein synthesis inhibitors and to cytochrome P-450 inhibitors. The findings are consistent with the hypothesis that the two enzyme activities are associated with different cytochrome P-450 moieties.  相似文献   
2.
Escherichia coli mutants lacking exonuclease III (xthA) are defective in the induction of heat-shock proteins upon severe heat-shock treatment (upshift from 30 to 50 degrees C) but not mild heat-shock treatment (upshift from 30 to 42 degrees C). We show that this defect is due to the xthA mutation by complementation. Furthermore, increasing the gene dosage of xthA+ prolongs the synthesis of heat shock proteins seen after a shift to 42 degrees C. Increasing the gene dosage of htpR+ partially suppresses the defect of xthA mutants in the synthesis of heat-shock proteins at 50 degrees C. When an xthA strain was incubated at 42 degrees C before a shift to 50 degrees C, it was then able to carry out the synthesis of heat-shock proteins at 50 degrees C.  相似文献   
3.
Because enzymes usually require distinct pH conditions for maximum activity, an approach of spontaneous pH shift of solution was devised to derive multiple reactions in a sequence. The two enzymes selected, horseradish peroxidase (HRP) and beta-galactosidase (GAL), had dissimilar optimal pH levels, i.e., 5.1 and 7.0, respectively. In a solution, HRP initially reacted at a lower pH range and the GAL reaction was consecutively carried out at a higher range in the presence of a third enzyme, urease, which caused an increase in pH. Under optimal conditions, the multiple system provided comparable performances with those of single reactions.  相似文献   
4.
OBJECTIVE--To determine the effects of patient''s sex and area''s material deprivation on utilisation rates of coronary catheterisation and angiography in the investigation of ischaemic heart disease. DESIGN--Retrospective analysis of routinely collected hospital statistics. SETTING--Acute hospitals throughout Northern Ireland. SUBJECTS--24,179 episodes of patients discharged from hospital with a primary diagnosis of ischaemic heart disease and 1270 episodes relating to patients with an underlying diagnosis of ischaemic heart disease who had either coronary catheterisation or angiography. MAIN OUTCOME MEASURES--Age standardised admission rates for heart disease and age standardised utilisation rates for catheterisation or angiography, or both, for 566 electoral wards ranked by Townsend "deprivation" scores. RESULTS--Catheterisation-angiography rates in men were over fivefold those of women, ranging from 85.5/100,000 v 16/100,000 in patients from "well off" areas to 123/100,000 v 22/100,000 for patients from deprived areas. After admission rates for heart disease were controlled for, the overall rate ratio for women was 0.48 (95% confidence interval 0.38 to 0.60). After differential admission rates for heart disease and other potential clinical confounders were controlled for, the investigation rates of patients from the least and most "deprived" areas were not significantly different (rate ratio 1.04 (0.87 to 1.25)). CONCLUSION--Although investigation rates were significantly lower in women than in men, further clinical data would be required before labelling this underutilisation as evidence of bias. There was no significant difference in invasive investigation rates for heart disease in areas of varying deprivation or affluence.  相似文献   
5.
Patients today demand more information about their treatment. Doctors, however, seem reluctant to cast aside ingrained habits of paternalism, believing they can best interpret therapeutic choices for their patients. Whether doctors can be more objective and effective than patients in interpreting the "probabilities" of medical evidence is open to question. On the other hand, the exercise of choice by patients may itself have a bearing on the probabilities of outcome. Involving patients more in making therapeutic choices is justified if doctors can present options in an unbiased and effective manner and if the process improves the outcome of the care delivered.  相似文献   
6.
Summary The unusual amino acid hypusine [N -(4-amino-2-hydroxybutyl)lysine] is a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology, eIF-4D). It is formed posttranslationally and exclusively in this protein in two consecutive enzymatic reactions, (i) modification of a single lysine residue of the eIF-5A precursor protein by the transfer of the 4-aminobutyl moiety of the polyamine spermidine to its-amino group to form the intermediate, deoxyhypusine [N -(4-aminobutyl)lysine] and (ii) subsequent hydroxylation of this intermediate to form hypusine. The amino acid sequences surrounding the hypusine residue are strictly conserved in all eukaryotic species examined, suggesting the fundamental importance of this amino acid throughout evolution. Hypusine is required for the activity of eIF-5Ain vitro. There is strong evidence that hypusine and eIF-5A are vital for eukaryotic cell proliferation. Inactivation of both of the eIF-5A genes is lethal in yeast and the hypusine modification appears to be a requirement for yeast survival (Schnier et al., 1991 [Mol Cell Biol 11: 3105–3114]; Wöhl et al., 1993 [Mol Gen Genet 241: 305–311]). Furthermore, inhibitors of either of the hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxyhypusine hydroxylase, exert strong anti-proliferative effects in mammalian cells, including many human cancer cell lines. These inhibitors hold potential as a new class of anticancer agents, targeting one specific eukaryotic cellular reaction, hypusine biosynthesis.  相似文献   
7.
Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana . Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis . Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis . The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1 , an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis .  相似文献   
8.
We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi. ERS-24 is localized to the ER and to the Golgi, and it is enriched in transport vesicles associated with these organelles.Newly formed transport vesicles have to be selectively targeted to their correct destinations, implying the existence of a set of compartment-specific proteins acting as unique receptor–ligand pairs. Such proteins have now been identified (Söllner et al., 1993a ; Rothman, 1994): one partner efficiently packaged into vesicles, termed a v-SNARE,1 and the other mainly localized to the target compartment, a t-SNARE. Cognate pairs of v- and t-SNAREs, capable of binding each other specifically, have been identified for the ER–Golgi transport step (Lian and Ferro-Novick, 1993; Søgaard et al., 1994), the Golgi–plasma membrane transport step (Aalto et al., 1993; Protopopov et al., 1993; Brennwald et al., 1994) in Saccharomyces cerevisiae, and regulated exocytosis in neuronal synapses (Söllner et al., 1993a ; for reviews see Scheller, 1995; Südhof, 1995). Additional components, like p115, rab proteins, and sec1 proteins, appear to regulate vesicle docking by controlling the assembly of SNARE complexes (Søgaard et al., 1994; Lian et al., 1994; Sapperstein et al., 1996; Hata et al., 1993; Pevsner et al., 1994).In contrast with vesicle docking, which requires compartment-specific components, the fusion of the two lipid bilayers uses a more general machinery derived, at least in part, from the cytosol (Rothman, 1994), which includes an ATPase, the N-ethylmaleimide–sensitive fusion protein (NSF) (Block et al., 1988; Malhotra et al., 1988), and soluble NSF attachment proteins (SNAPs) (Clary et al., 1990; Clary and Rothman, 1990; Whiteheart et al., 1993). Only the assembled v–t-SNARE complex provides high affinity sites for the consecutive binding of three SNAPs (Söllner et al., 1993b ; Hayashi et al., 1995) and NSF. When NSF is inactivated in vivo, v–t-SNARE complexes accumulate, confirming that NSF is needed for fusion after stable docking (Søgaard et al., 1994).The complex of SNAREs, SNAPs, and NSF can be isolated from detergent extracts of cellular membranes in the presence of ATPγS, or in the presence of ATP but in the absence of Mg2+, and sediments at ∼20 Svedberg (20S particle) (Wilson et al., 1992). In the presence of MgATP, the ATPase of NSF disassembles the v–t-SNARE complex and also releases SNAPs. It seems likely that this step somehow initiates fusion.To better understand vesicle flow patterns within cells, it is clearly of interest to identify new SNARE proteins. Presently, the most complete inventory is in yeast, but immunolocalization is difficult in yeast compared with animal cells, and many steps in protein transport have been reconstituted in animal extracts (Rothman, 1992) that have not yet been developed in yeast. Therefore, it is important to create an inventory of SNARE proteins in animal cells. The most unambiguous and direct method for isolating new SNAREs is to exploit their ability to assemble together with SNAPs and NSF into 20S particles and to disassemble into subunits when NSF hydrolyzes ATP. Similar approaches have already been successfully used to isolate new SNAREs implicated in ER to Golgi (Søgaard et al., 1994) and intra-Golgi transport (Nagahama et al., 1996), in addition to the original discovery of SNAREs in the context of neurotransmission (Söllner et al., 1993a ).Using this method, we now report the isolation and detailed characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE that is localized to the ER and Golgi. ERS-24 is found in transport vesicles associated with the transitional areas of the ER and with the rims of Golgi cisternae, suggesting a role for ERS-24 in vesicular transport between these two compartments.  相似文献   
9.
The concept of a competitive enzyme immunoassay that utilizes simultaneously the bound and the free analyte-enzyme conjugate (heterobifunctional conjugate) for signal generation in response to varying analyte concentrations in samples has been investigated. Two antigenic sites of the heterobifunctional conjugate are used in the assay for binding to immunoglobulins: the analyte derivative binds to an immobilized antibody, Ab(1), and the enzyme component binds to a spatially separated immobilized antibody, Ab(2). The analytical system is set up such that in the absence of analyte, the conjugate is predominantly bound in the compartment that contains Ab(1). With increasing concentration of native analyte in samples, an increasing concentration of native analyte in samples, an increasing amount of conjugate migrates to the second compartment that contains Ab(2). The enzyme bound in each compartment is used for signal generation. Mathematical models have been developed to determine the optimal conditions and to predict the performance of such dual-antibody systems. The theoretical predictions are supported by experimental results. The dual-antibody system has been compared with a conventional competitive enzyme immunoassay using the same reagents.  相似文献   
10.
The level of ventilation attained and breathing patterns adopted during activity have important implications for the distribution and deposition of particles that are inhaled. However, breathing patterns and levels of ventilation adopted during specific physical activities are unknown. We used a noninvasive means of measuring ventilation in subjects performing a variety of activities (bicycling, arm ergometry, lifting, and pulling) during unencumbered (no mouthpiece) breathing and while breathing through a mouthpiece. Minute ventilation (VE), tidal volume (VT), inspiratory time (TI), and total breathing cycle time (TT) were measured initially both spirometrically and from body surface displacements. When a mouthpiece was used, VE and breathing patterns were significantly altered during all activities such that VE, VT, and TT increased by 16, 34, and 20%, respectively. This mouthpiece effect was attenuated at the higher levels of VE. A task dependency of breathing pattern was also noted such that there was much greater variability of VT and TI for a given VE during the lifting activity compared with bicycling (coefficient of variation for VT of 0.39 +/- 0.09 vs. 0.20 +/- 0.07, P less than 0.01; and for TI of 0.38 +/- 0.08 vs. 0.21 +/- 0.08, P less than 0.01). We conclude that a mouthpiece significantly alters breathing pattern during varied types and intensities of activities, and breathing patterns may differ significantly from one activity to another. When the total dose of particulates inhaled in the lung are assessed, the mouthpiece effect and activity effect on breathing pattern must be considered.  相似文献   
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