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81.
Sen S Jaakola VP Heimo H Engström M Larjomaa P Scheinin M Lundstrom K Goldman A 《Protein expression and purification》2003,32(2):265-275
The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology. 相似文献
82.
The purpose of this research was to improve the stability of carbamazepine (CBZ) bulk powder under high humidity by surface
modification. The surface-modified anhydrates of CBZ were obtained in a specially designed surface modification apparatus
at 60°C via the adsorption of n-butanol, and powder x-ray diffraction, Fourier-Transformed Infrared spectra, and differential
scanning calorimetry were used to determine the crystalline characteristics of the samples. The hydration process of intact
and surface-modified CBZ anhydrate at 97% relative humidity (RH) and 40±1°C was automatically monitored by using isothermal
microcalorimetry (IMC). The dissolution test for surface-modified samples (20 mg) was performed in 900 mL of distilled water
at 37±0.5°C with stirring by a paddle at 100 rpm as in the Japanese Pharmacopoeia XIII. The heat flow profiles of hydration
of intact and surface-modified CBZ anhydrates at 97% RH by using IMC profiles showed a maximum peak at around 10 hours and
45 hours after 0 and 10 hours of induction, respectively. The result indicated that hydration of CBZ anhydrate was completely
inhibited at the initial stage by surface modification of n-butanol and thereafter transformed into dihydrate. The hydration
of surface-modified samples followed a 2-dimensional phase boundary process with an induction period (IP). The IP of intact and surface-modified samples decreased with increase of the reaction temperature, and the hydration rate
constant (k) increased with increase of the temperature. The crystal growth rate constants of nuclei of the intact sample were significantly
larger than the surface-modified samples at each temperature. The activation energy (E) of nuclei formation and crystal growth process for hydration of surface-modified CBZ anhydrate were evaluated to be 20.1
and 32.5 kJ/mol, respectively, from Arrhenius plots, but the Es of intact anhydrate were 56.3 and 26.8 kJ/mol, respectively. The dissolution profiles showed that the surface-modified sample
dissolved faster than the intact sample at the initial stage. The dissolution kinetics were analyzed based on the Hixon-Crowell
equation, and the dissolution rate constants for intact and surface-modified anhydrates were found to be 0.0102±0.008 mg1/3 min−1 and 0.1442±0.0482 mg1/3·min−1. The surface-modified anhydrate powders were more stable than the nonmodified samples under high humidity and showed resistance
against moisture. However, surface modification induced rapid dissolution in water compared to the control. 相似文献
83.
Olli-Lähdesmäki T Scheinin M Pohjanoksa K Kallio J 《European journal of cell biology》2003,82(5):231-239
Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha2A- and alpha2B-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha2A- and alpha2B-AR were internalized into partly distinct intracellular vesicles (Olli-L?hdesm?ki et al., J. Neurosci. 19, 9281-9288, 1999). In this paper, the extent of alpha2-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human alpha2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha2B-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha2A-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha2A- and alpha2B-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy. 相似文献
84.
The toxicity and effects on protein synthesis of the phthalate esters diethyl phthalate (DEP) and di(2-ethylhexyl) phthalate (DEHP) was studied in radish seedlings (Raphanus sativus cv. Kööpenhaminan tori). Phthalate esters are a class of commercially important compounds used mainly as plasticizers in high molecular-weight polymers such as many plastics. They can enter soil through various routes and can affect plant growth and development. First the effect of DEP and DEHP on the growth of radish seedlings was determined in an aqueous medium. It was found that DEP, but not DEHP, caused retardation of growth in radish. A further investigation on protein synthesis during DEP-stress was executed by in vivo protein labeling combined with two-dimensional gel electrophoresis (2D-PAGE). For comparisons with known stress-induced proteins a similar experiment was done with heat shock, and the induced heat shock proteins (HSPs) were compared with those of DEP-stress. The results showed that certain HSPs can be used as an indicator of DEP-stress, although the synthesis of most HSPs was not affected by DEP. DEP also elicited the synthesis of numerous proteins found only in DEP-treated roots. The toxic effect of phthalate esters and the roles of the induced proteins are discussed. 相似文献
85.
The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS. 相似文献
86.
Ueda A Nagase S Yokoyama H Tada M Noda H Ohya H Kamada H Hirayama A Koyama A 《Molecular and cellular biochemistry》2003,244(1-2):119-124
Spin probing methods using an electron spin resonance (ESR) spectrometer are used extensively and bring us a lot of information about in vivo redox mechanisms. However, the in vivo reducing mechanisms of exogenous nitroxide radicals, which serve as typical spin probing reagents are not clear. To clarify this, we examined the sequential kinetics of a spin probe, 4-hydroxy 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) in the in vivo organs, tissue homogenates and subcellular fractions of kidney and liver using an in vivo and X-band ESR spectrometers. As a parameter of reducing activity, we calculated the half-life of TEMPOL from the decay curve of ESR signal intensity. The half-life of TEMPOL in the whole organs and homogenates of the kidney was significantly shorter than that of the liver, this indicates that the kidney has more reducing activity against TEMPOL as compared to the liver. Subcellular fractional studies revealed that this reducing activity of the kidney mainly exists in the mitochondria. Contrarily, in addition to reduction in the mitochondria, TEMPOL in the liver was reduced by the microsome and cytosol. 相似文献
87.
Real time observation of reaction kinetics is one of the key features of the newly developed microparticle based two-photon excitation fluorescence immunoassay system (TPX). By observing binding reactions at the surface of individual microparticles during the incubation of an assay, the binding constants of an assay become apparent. This paper describes the use of the new system in quantifying the reaction parameters of human thyroid stimulating hormone (hTSH) assay. A mechanistic reaction model for the assay is presented. The reaction model is further shown to precisely predict the behaviour of the assay kinetics over a wide range of analyte concentrations. 相似文献
88.
In eight hagfish species, it is known that chromosome elimination occurs during early embryogenesis, and some highly repetitive
DNA families, restricted to germ cells, have been isolated. One of these families, ``EEEo2,' has been isolated as DNA fragments
by restriction enzyme analyses from Eptatretus okinoseanus and E. cirrhatus. In this study, EEEo2 sequences were isolated from germline DNA in E. burgeri, Paramyxine sheni, and P. atami using PCR methods. Sequence analysis revealed that these sequences are intraspecifically homogeneous, except in E. burgeri, and are interspecifically conserved with heterogeneity. The intraspecific sequence variability tends to decrease as the copy
number increases. These results indicate that EEEo2 has evolved in a concerted manner. Moreover, an ancestral repeating motif
consisting of triplicate subrepeats was deduced. These results suggest that EEEo2 arose as an initial amplification of this
subrepeat and has evolved by saltatory replication. Phylogenetic analyses suggested the possibility that EEEo2 in E. okinoseanus and E. cirrhatus has been subjected to strong homogenizing forces for concerted evolution, whereas the force is weak in E. burgeri. In addition, EEEo2 in P. sheni and P. atami appear to have been incompletely subjected to these forces. Chromosomal in situ hybridization experiments revealed that EEEo2
sequences were located along almost their entire length of several heterochromatic chromosomes that are restricted to germ
cells. These chromosomes are disposed to form a secondary association during the first meiotic metaphases, except in P. sheni. This chromosomal distribution may promote a concerted mode of sequence evolution in both nonhomologous chromosomes and homologous
chromosomes and reflect the differential driving forces between species.
Received: 17 April 1999 / Accepted: 10 September 1999 相似文献
89.
In chick limb buds, mesenchymal cells of the progress zone (PZ-cells) at different developmental stages segregate one from the other in mixed cell cultures, suggesting they have different cell affinity. In order to learn the possible roles of such differences in the cells, two heterotypic leg PZ-cell populations (cells from stages 25/26 and 20/21) in vitro were juxtaposed to allow them to form the boundary. A method with double cylindrical columns was used to make adjoining monolayer cell cultures. It was shown that heterotypic juxtaposition produced two chondrogenic patterns along the boundary: aggregates of chondrocytes formed by stage 20/21 PZ-cells and a chondrocyte-free band formed by those at stage 25/26. Juxtaposition of PZ-cells and proximal cells also formed these patterns, while that between cells from anterior and posterior PZ formed indistinct patterns along the boundary. Homotypic PZ-cell juxtaposition did not produce these patterns. The results suggest that different cell affinity has a role in the segmentation of cartilage patterns at a point along the proximodistal axis, as well as a role in retaining cells in one area so as not to be recruited to other condensation areas. 相似文献
90.
Different metabolic responses of human brown adipose tissue to activation by cold and insulin 总被引:1,自引:0,他引:1
Orava J Nuutila P Lidell ME Oikonen V Noponen T Viljanen T Scheinin M Taittonen M Niemi T Enerbäck S Virtanen KA 《Cell metabolism》2011,14(2):272-279
We investigated the metabolism of human brown adipose tissue (BAT) in healthy subjects by determining its cold-induced and insulin-stimulated glucose uptake and blood flow (perfusion) using positron emission tomography (PET) combined with computed tomography (CT). Second, we assessed gene expression in human BAT and white adipose tissue (WAT). Glucose uptake was induced 12-fold in BAT by cold, accompanied by doubling of perfusion. We found a positive association between whole-body energy expenditure and BAT perfusion. Insulin enhanced glucose uptake 5-fold in BAT independently of its perfusion, while the effect on WAT was weaker. The gene expression level of insulin-sensitive glucose transporter GLUT4 was also higher in BAT as compared to WAT. In conclusion, BAT appears to be differently activated by insulin and cold; in response to insulin, BAT displays high glucose uptake without increased perfusion, but when activated by cold, it dissipates energy in a perfusion-dependent manner. 相似文献