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971.
Gordana ?or?evi? Koviljka Matu?an Ilija? Ita Had?isejdi? Anton Mari?i? Bla?enka Grahovac Nives Jonji? 《Journal of biomedical science》2012,19(1):40
Background
The role of epidermal growth factor (EGF) and its receptor (EGFR) in the pathogenesis and progression of various malignant tumors has long been known, but there is still disagreement concerning prognostic significance of EGFR expression in clear cell renal cell carcinoma (CCRCC). The present study was designed to analyze more objectively the protein EGFR expression in CCRCC and to compare its value with EGFR gene copy number changes and clinicopathologic characteristics including patient survival.Methods
The protein EGFR expression was analyzed immunohistochemically on 94 CCRCC, and gene copy number alterations of EGFR by FISH analysis on 41 CCRCC selected according to distinct membrane EGFR staining.Results
Membrane EGFR expression in tumor cells was heterogeneous with respect to the proportion of positive cells and staining intensity. FISH analysis did not reveal EGFR gene amplification, while polysomy of chromosome 7 found in 41% was associated with higher EGFR membrane expression. Moreover, EGFR overexpression was associated with a higher nuclear grade, larger tumor size and shorter patient''s survival, while there was no connection with pathological stage.Conclusion
In conclusion, the protein expression of EGFR had an impact on prognosis in patients with CCRCC, while an increased copy number of chromosome 7 could be the possible reason for EGFR protein overexpression in the absence of gene amplification. 相似文献972.
Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels. 相似文献
973.
Ryuichiro Suzuki Kazue Terasawa Keitarou Kimura Zui Fujimoto Mitsuru Momma Mikihiko Kobayashi Atsuo Kimura Kazumi Funane 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(7):919-924
Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the kcat/KM value of CITase-598K was approximately two times higher (32.2 s− 1 mM− 1) than that of CITase-T3040 (17.8 s− 1 mM− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes. 相似文献
974.
Tatiana F.R. CostaFlavia C.G. dos Reis Ana Paula C.A. Lima 《Biochimica et Biophysica Acta - Proteins and Proteomics》2012,1824(3):493-501
The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the Km, but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25 °C but concealed at 37 °C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25 °C, but not at 37 °C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation. 相似文献
975.
The P2X7 receptor is widely recognized to mediate the proinflammatory effects of extracellular ATP. However this receptor in the absence of ATP may have a function unrelated to inflammation. Our data show that P2X7 expressed on the surface of monocyte/macrophages or on epithelial HEK-293 cells greatly augments the engulfment of latex beads and live and heat-killed bacteria by effector phagocyte in the absence of ATP and serum. The expression of P2X7 on the effector also confers the ability to phagocytose apoptotic target cells and an accumulation of P2X7 can be seen at the attachment point to the target. Activation of the P2X7 receptor by ATP causes a slow dissociation (over 10–15 min) of nonmuscle myosin from the P2X7 membrane complex and abolishes further P2X7-mediated phagocytosis of these targets. The recent crystal structure of the homologous zebrafish P2X4 receptor shows an exposed “nose” of the ectodomain (residues 115–162) which contains three of the five disulfide bonds conserved in all P2X receptors. Three short biotin-labeled peptides mimicking sequence of this exposed region bound to apoptotic target cells but not to either viable cells or to other target particles. All three peptides contained one or two cysteine residues and their replacement by alanine abolished peptide binding. These data implicate thiol-disulfide exchange reactions in the initial tethering of apoptotic cells to macrophage and establish P2X7 as one of the scavenger receptors involved in the recognition and removal of apoptotic cells in the absence of extracellular ATP and serum. 相似文献
976.
8‐Hydroxyquinoline‐7‐carboxaldehyde (8‐HQ‐7‐CA), Schiff‐base ligand 8‐hydroxyquinoline‐7‐carboxaldehyde benzoylhydrazone, and binuclear complexes [LnL(NO3)(H2O)2]2 were prepared from the ligand and equivalent molar amounts of Ln(NO3)?6 H2O (Ln=La3+, Nd3+, Sm3+, Eu3+, Gd3+, Dy3+, Ho3+, Er3+, Yb3+, resp.). Ligand acts as dibasic tetradentates, binding to LnIII through the phenolate O‐atom, N‐atom of quinolinato unit, and C?N and ? O? C?N? groups of the benzoylhydrazine side chain. Dimerization of this monomeric unit occurs through the phenolate O‐atoms leading to a central four‐membered (LnO)2 ring. Ligand and all of the LnIII complexes can strongly bind to CT‐DNA through intercalation with the binding constants at 105–106 M ?1. Moreover, ligand and all of the LnIII complexes have strong abilities of scavenging effects for hydroxyl (HO.) radicals. Both the antioxidation and DNA‐binding properties of LnIII complexes are much better than that of ligand. 相似文献
977.
Daniel de los Cobos‐Vasconcelos Nora Ruiz‐Ordaz Juvencio Galíndez‐Mayer Héctor Poggi‐Varaldo Cleotilde Juàrez‐Ramírez López‐Muñoz Aarón 《Engineering in Life Science》2012,12(1):39-48
The biodegradation of the sulfonated azo dyes, Acid Orange 7 (AO7) and Acid Red 88 (AR88), by a bacterial consortium isolated from water and soil samples obtained from sites receiving discharges from textile industries, was evaluated. For a better removal of azo dyes and their biodegradation byproducts, an aerobically operated two‐stage rectangular packed‐bed biofilm reactor (2S‐RPBR) was constructed. Because the consortium's metabolic activity is affected by oxygen, the effect of the interstitial air flow rate QGI on 2S‐RPBR's zonal values of the oxygen mass transfer coefficient kLa was estimated. In the operational conditions probed in the bioreactor, the kLa values varied from 3 to 60 h?1, which roughly correspond to volumetric oxygen transfer rates, dcL/dt, ranging from 20 to 375 mg O2 L?1h?1. Complete biodegradation of azo dyes was attained at loading rates BV,AZ up to 40 mg L?1d?1. At higher BV,AZ values (80 mg L?1 d?1), dye decolorization and biodegradation of the intermediaries 4‐amino‐naphthalenesulphonic acid (4‐ANS) and 1‐amino‐2‐naphthol (1‐A2N) was almost complete. However, a diminution in COD and TOC removal efficiencies was observed in correspondence to the 4‐aminobenzenesulfonic acid (4‐ABS) accumulation in the bioreactor. Although the oxygen transport rate improved the azo dye mineralization, the results suggest that the removal efficiency of azo dyes was affected by biofilm detachment at relatively high QGI and BV,AZ values. After 225 days of continuous operation of the 2S‐RFBR, eight bacterial strains were isolated from the biofilm attached to the porous support. The identified genera were: Arthrobacter, Variovorax, Agrococcus, Sphingomonas, Sphingopyxis, Methylobacterium, Mesorhizobium, and Microbacterium. 相似文献
978.
979.
Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called autophagosome. SNAREs proteins are key molecules of the vesicle fusion machinery. Our results indicate that in a mammalian tumor cell line a subset of VAMP7 (V-SNARE)-positive vacuoles colocalize with LC3 at the cell periphery (focal adhesions) upon starvation. The re-distribution of VAMP7 positive structures is a microtubule-dependent event, with the participation of the motor protein KIF5 and the RAB7 effector RILP. Interestingly, most of the VAMP7-labeled vesicles were loaded with ATP. Moreover, in cells subjected to starvation, these structures fuse with the plasma membrane to release the nucleotide to the extracellular medium. Summarizing, our results show the molecular components involved in the release of ATP to extracellular space, which is recognized as an important autocrine/paracrine signal molecule that participates in the regulation of several cellular functions such as immunogenicity of cancer cell death or inflammation 相似文献
980.
Johan A. Stenberg 《Insect Science》2012,19(5):604-608
Abstract The parasitoid Asecodes mento (Walker, 1839) (Hymenoptera: Eulophidae) is the most important biocontrol agent of the strawberry leaf beetle Galerucella tenella (L.) (Coleoptera: Chrysomelidae) in northern Europe. Here, I investigated whether natural parasitism in organic strawberry plantations was affected by the presence of the alternative host plant meadowsweet (Filipendula ulmaria), and whether parasitism rates differed between plantations of different ages (6 to 79 years). I also investigated whether parasitoid brood size, body size and sex ratio differed between the two host plants in the field. Parasitism was very low (0%) in newly established plantations and increased to a plateau (~40%) in fields where strawberries had been grown for approximately 20 years or longer. Such an extended colonization process is unacceptable for commercial growers. It would thus be desirable to find a method to catalyze parasitoid population buildup in young plantations. Parasitoid brood sizes were larger in beetles collected from meadowsweet, while body size and sex ratio did not differ between parasitoids collected from the two plants. These findings suggest that meadowsweet can export parasitoids to neighboring strawberry fields. Although this is a possibility, I did not find any significant differences in parasitism rates between isolated strawberry fields and fields adjacent to meadowsweet stands, indicating that effects of local vegetation are small on parasitism rates. Releasing parasitoids in newly established strawberry plantations may be a better strategy for quickly obtaining high parasitism than intercropping with meadowsweet. 相似文献