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171.
Gresz V Kwon TH Gong H Agre P Steward MC King LS Nielsen S 《American journal of physiology. Gastrointestinal and liver physiology》2004,287(1):G151-G161
In vitro studies of cultured salivary gland cells and gland slices have indicated that there may be regulated translocation of aquaporin (AQP)-5 between the apical plasma membrane and intracellular compartments of the secretory cells. However, it remains unknown whether AQP-5 in salivary glands is subject to regulated trafficking in vivo. To examine this possibility, we have investigated the subcellular localization of AQP-5 in rat parotid and submandibular glands fixed in vivo under conditions of stimulated or inhibited salivary secretion. Immunofluorescence and immunoelectron microscopy was used to determine the subcellular distribution of AQP-5 in control conditions following the stimulation of secretion with pilocarpine (a muscarinic agonist) or epinephrine (an alpha-adrenoceptor agonist) or during inhibition of basal secretion with atropine (a muscarinic antagonist) or phentolamine (an alpha-adrenoceptor antagonist). Under control conditions, >90% of AQP-5 was associated with the apical plasma membrane of acinar and intercalated duct cells, with only rare gold particles associated with intracellular membrane domains. Pilocarpine treatment dramatically increased saliva production but had no discernible effect on AQP-5 distribution. However, the increased salivary secretion was associated with luminal dilation and the appearance of a markedly punctate AQP-5 labeling pattern due to clustering of AQP-5 at the microvilli (especially evident in the parotid gland) after 10 min of drug injection. No changes in the subcellular localization of AQP-5 were seen in response to epinephrine, atropine, or phentolamine treatment compared with control tissues. Thus AQP-5 is localized predominantly in the apical plasma membrane under control conditions, and neither the onset nor the cessation of secretion is associated in vivo with any significant short-term translocation of AQP-5 between intracellular structures and the apical plasma membrane. 相似文献
172.
Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain 总被引:7,自引:0,他引:7
Mammalian cells control their membrane composition by regulating the vesicular transport of membrane-bound sterol regulatory element binding proteins (SREBPs) from endoplasmic reticulum (ER) to Golgi. Transport is blocked by cholesterol, which triggers SCAP, the SREBP escort protein, to bind to Insigs, which are ER retention proteins. The cholesterol trigger mechanism is unknown. Using recombinant SCAP purified in detergent, we show that cholesterol acts by binding with high affinity and specificity to the 767 amino acid octahelical membrane region of SCAP. This octahelical region contains a conserved pentahelical sterol-sensing domain found in six other polytopic membrane proteins. We show that the membrane domain of SCAP is a tetramer and that cholesterol binding is inhibited by cationic amphiphiles, raising the possibility of allosteric regulation by positively charged phospholipids. The current studies show that cells control their cholesterol content through receptor-ligand interactions and not through changes in the physical properties of the membrane. 相似文献
173.
Lee J Nam S Hwang SB Hong M Kwon JY Joeng KS Im SH Shim J Park MC 《Journal of biochemistry and molecular biology》2004,37(1):107-113
Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology. 相似文献
174.
Jae-Hwa?Lee Nguyen-Hoang?Loc Tae-Ho?Kwon Moon-Sik?YangEmail author 《Biotechnology and Bioprocess Engineering》2004,9(1):12-16
Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems
(ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene
glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium
phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system,
hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andK
hGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase
with a yield of 45.66% andK
hGM-CSF of 7.64 after 2 h of incubation at room temperature. 相似文献
175.
Park SY Ryu SH Jang IC Kwon SY Kim JG Kwak SS 《Molecular genetics and genomics : MGG》2004,271(3):339-346
A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1 , was isolated from cell cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweetpotato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 M), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 M) or exposure to high temperature (37°C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen (Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.Communicated by G. Jürgens 相似文献
176.
Escharectomy has been shown to improve the survival rates and the outcomes in burns. This observational study was conducted to assess the role of escharectomy on the inflammatory mediators in major burns. Seventeen ASA physical status II or status III adult surviving major burn patients were recruited. When the escharectomy was scheduled, a series of blood samples was obtained at -3 and -1 days preoperation, and +1 and +3 postoperation. The changing levels of endotoxin, cytokines, and adhesion molecules were measured with a quantitative sandwich immunoassay. Extensive escharectomy did not appear to have any significant impact on the levels of tumor necrosis factor alpha, interleukin-10, soluble intracellular adhesion molecule-1 and soluble vascular adhesion molecule-1. Meanwhile, endotoxin and E-selectin were significantly decreased after escharectomy. Escharectomy appeared to have a limited immunomodulatory effect on the inflammatory mediators in systemic inflammatory responses induced by major burns. This is probably related to the timing and extent of surgery, and the complex nature of burn-related inflammation. 相似文献
177.
Kim DW Yoon CS Eum WS Lee BR An JJ Lee SH Lee SR Ahn JY Kwon OS Kang TC Won MH Cho SW Lee KS Park J Choi SY 《Molecules and cells》2004,18(3):314-319
gamma-Aminobutyrate transaminase (GABA-T), a key enzyme of the GABA shunt, converts the major inhibitory neurotransmitter, GABA, to succinic semialdehyde. Although GABA-T is a pivotal factor implicated in the pathogenesis of various neurological disorders, its function remains to be elucidated. In an effort to clarify the structural and functional roles of specific lysyl residue in human brain GABA-T, we constructed human brain GABA-T mutants, in which the lysyl residue at position 357 was mutated to various amino acids including asparagine (K357N). The purified mutant GABA-T enzymes displayed neither catalytic activity nor absorption bands at 330 and 415 nm that are characteristic of pyridoxal-5'-phosphate (PLP) covalently linked to the protein. The wild type apoenzyme reconstituted with exogenous PLP had catalytic activity, while the mutant apoenzymes did not. These results indicate that lysine 357 is essential for catalytic function, and is involved in binding PLP at the active site. 相似文献
178.
Changes in parvalbumin immunoreactivity in the parietofrontal cortex after transient forebrain ischemia in the Mongolian gerbil 总被引:1,自引:0,他引:1
Hwang IK Yoo KY Kim DS Jung JY Kim KS Park JK Kwon YG Lee JY Kang TC Won MH 《Molecules and cells》2004,17(2):304-308
We investigated the changes in parvalbumin (PV)-immunoreactive (IR) neurons in the parietofrontal cortex after transient forebrain ischemia. In the sham-operated group, PV-IR neurons were present in all layers of the parietofrontal cortex except layer I. Shortly after ischemia the number of PV-IR neurons in layer II/III first increased, and then declined dramatically 12 h after ischemic insult, followed by a second increase after 2 days. At this time the PV immunoreactivity was very weak and only present in the peripheral neuronal cytoplasm. The reversible increase in the number of PV-IR neurons and in the level of their immunoreactivity could result from a transient ischemia-induced increase in intracellular calcium. This pattern of expression was particularly pronounced in layer II/III of the parietofrontal cortex, suggesting that these neurons are especially\susceptible to ischemic insult. 相似文献
179.
180.
Yoon CS Kim DW Jang SH Lee BR Choi HS Choi SH Kim SY An JJ Kwon OS Kang TC Won MH Cho SW Lee KS Park J Eum WS Choi SY 《Molecules and cells》2004,18(2):214-219
Gamma-aminobutyrate transaminase (GABA-T), a key homodimeric enzyme of the GABA shunt, converts the major inhibitory neurotransmitter GABA to succinic semialdehyde. We previously overexpressed, purified and characterized human brain GABA-T. To identify the structural and functional roles of the cysteinyl residue at position 321, we constructed various GABA-T mutants by site-directed mutagenesis. The purified wild type GABA-T enzyme was enzymatically active, whereas the mutant enzymes were inactive. Reaction of 1.5 sulfhydryl groups per wild type dimer with 5,5 cent-dithiobis-2-nitrobenzoic acid (DTNB) produced about 95% loss of activity. No reactive -SH groups were detected in the mutant enzymes. Wild type GABA-T, but not the mutants, existed as an oligomeric species of Mr = 100,000 that was dissociable by 2-mercaptoethanol. These results suggest that the Cys321 residue is essential for the catalytic function of GABA-T, and that it is involved in the formation of a disulfide link between two monomers of human brain GABA-T. 相似文献