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71.
TRPM2 is a tetrameric Ca2+-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca2+, but the mechanism and the binding sites for Ca2+ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca2+ dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca2+ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca2+ activated ∼50-pS nonselective cation channels; K1/2 for ADPR was ∼1 µM at saturating Ca2+. Intracellular Ca2+ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca2+) reveals that Ca2+ activation is a consequence of tighter binding of Ca2+ in the open rather than in the closed channel conformation. Four Ca2+ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 106-fold activation. Experiments in the presence of 1 mM of free Ca2+ on the extracellular side clearly show that closed channels do not sense extracellular Ca2+, but once channels have opened Ca2+ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca2+-free wash of the intracellular channel surface. This effect of extracellular Ca2+ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca2+ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca2+ likely triggers prolonged, self-sustained TRPM2 activity. 相似文献
72.
Miko E Margitai Z Czimmerer Z Várkonyi I Dezso B Lányi A Bacsó Z Scholtz B 《FEBS letters》2011,585(8):1191-1196
Despite intensive efforts to improve therapies, small cell lung cancer (SCLC) still has a dismal median survival of 18 months. Since miR-126 is under-expressed in the majority of SCLC tumors, we investigated the effect of miR-126 overexpression on the proliferation and cell cycle distribution of H69 cells. Our results demonstrate that miR-126 inhibits proliferation of H69 cells, by delaying the cells in the G1 phase. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of mir-126, has the same effect on H69 cells. We also show for the first time that SLC7A5 is a direct target of miR-126. 相似文献
73.
Ladányi A Kiss J Mohos A Somlai B Liszkay G Gilde K Fejös Z Gaudi I Dobos J Tímár J 《Cancer immunology, immunotherapy : CII》2011,60(12):1729-1738
Studies on the prognostic importance of tumor-infiltrating lymphocytes have mainly focused on T cells, while little is known
about the role of tumor-infiltrating B lymphocytes. We investigated the prevalence of CD20+ B cells by immunohistochemistry in primary melanoma samples of 106 patients and analyzed in relation to clinicopathological
parameters and patients’ survival. The majority of samples contained a significant amount of B lymphocytes, predominantly
dispersed in the stroma surrounding tumor deposits (mean peritumoral and intratumoral densities: 178.7 ± 156.1 vs. 4.9 ± 6.9
cells/mm2, respectively). B cells organized in follicle-like aggregates were also observed in 26% of the samples. B-cell density correlated
with that of activated (CD25+ or OX40+) T lymphocytes. Infiltration by CD20+ lymphocytes did not correlate with tumor thickness, while the presence of B-cell aggregates was observed more frequently
in thick melanomas. On the other hand, B-cell infiltration was more pronounced in nonmetastatic or lymph node metastatic tumors,
compared to visceral metastatic ones. Accordingly, high number of these cells provided significant survival advantage (P = 0.0391 and P = 0.0136 for intra- and peritumoral infiltration, respectively). Furthermore, combination of peritumoral B-cell density with
the number of activated T lymphocytes identified patient subgroups with different disease outcome, which was most favorable
in the case of high density, while very poor in the case of low density of both cell types. Multivariate survival analysis
identified tumor thickness and CD20+/OX40+ cell density combination as significant independent prognostic factors. Taken together, our results show correlation between
low number of CD20+ B lymphocytes and melanoma progression, indicating a possible role of tumor-infiltrating B cells in antitumoral immune response.
It was also reflected in better outcome of the disease since the density of B lymphocytes alone as well as in combination
with that of activated T cells proved of prognostic importance in patients with malignant melanoma. 相似文献
74.
Piršelová B Kuna R Libantová J Moravčíková J Matušíková I 《Molecular biology reports》2011,38(5):3437-3446
Defense responses against cadmium, arsenic and lead were compared in two crop plants such as the monocotyledonous maize (Zea mays cv. Quintal) and dicotyledonous soybean (Glycine max cv. Korada). The applied metals caused root growth retardation, membrane damage and subsequent loss of cell viability, while
enhanced H2O2 generation, lipid peroxidation and lignification were detected with respect to corresponding controls. The measured data
suggest that soybean was in general more tolerant to tested doses of metals and showed more pronounced defense responses than
maize. Concurrently, the total activity of β-1,3-glucanases, a subgroup of so called pathogenesis-related defense proteins,
was comparable in root extracts of both plant types. Though in a view of previous comparative genome approaches the β-1,3-glucanases
do not mirror the differences in the cell wall structure and architecture between the monocots and dicots, we show that in
both plant types they clearly respond to metal stress. Accumulation patterns of different glucanase isoforms upon exposure
to tested metals indicate that they do contribute to plant defense mechanisms during exposure to heavy metals and their biological
role is more complex than expected. 相似文献
75.
Vig A Ohmacht R Jámbor E Bugyi B Nyitrai M Hild G 《European biophysics journal : EBJ》2011,40(5):619-626
During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy
nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics
of P
i
release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique
was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P
i
release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by
release of P
i
from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples
revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P
i
. The effect of both toxins can be sufficiently explained by local and mechanical blockade of P
i
dissociation. 相似文献
76.
Valsecchi F Monge C Forkink M de Groof AJ Benard G Rossignol R Swarts HG van Emst-de Vries SE Rodenburg RJ Calvaruso MA Nijtmans LG Heeman B Roestenberg P Wieringa B Smeitink JA Koopman WJ Willems PH 《Biochimica et biophysica acta》2012,1817(10):1925-1936
Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012). 相似文献
77.
Ultrastructural studies were conducted on asexual developmental stages of C. parvum in the ileal fragment of the intestine of 10-11 day old rats experimentally infected with oocysts isolated from calf feces. A young trophozoite is covered with the typical trimembranous apicomplexan pellicle. As the parasite grows, the inner complex of its apical pellicle, facing the host enterocyte, is seen to reduce up to a unit membrane to make a complex multimembranous "feeding organelle" which is in contact with a thick electron dense band bordering the host-parasite interface. It looks likely that no micropores or any other feeding structures exist in the parasite. Unlike, the opposite body part of the trophozoite, facing the lumen of the intestine, preserves its trimembranous pellicle. Two merozoite generations were followed. In addition to numerous ribosomes, rhoptries, micronemes, and trimembranous pellicle, subpellicular microtubules were observed in the segmenting merozoites. The merogony follows the pattern of ectomeric schizogony. However, no details of nuclear division were detected. The whole cytoplasm of the mother meront is completely used up for the merozoite formation without any residual mass to be left. 相似文献
78.
膀胱移行细胞和移行细胞癌细胞内β-葡萄糖醛酸酶免疫电镜比较研究 总被引:1,自引:0,他引:1
β-葡萄糖醛酸酶(β-Glucuronidase,简称β-G)在正常人体组织匀浆和体液中含量很低,本实验采用胶体金标记,免疫电镜技术,进行了人体正常移行细胞与移行细胞癌细胞内β-G定位研究,实验结果表明,β-G存在于移行细胞和移行细胞癌细胞中的内质网、溶酶体内,同时观察到癌细胞中标记β-G的金颗粒数量多于正常移行细胞中金颗粒的数量,本实验结果可能对于移行细胞癌的早期发现、早期诊断提供了新的依据。 相似文献
79.
Measurements of tolerance to the addition of benzaldehyde were carried out with six yeast species:Hansenula anomala, Brettanomyces vini Peynaud et Domercq, strain X,Saccharomyces carlsbergensis, Saccharomyces cerevisiae R XII,Saccharomyces ellipsoideus andTorula utilis. The techniques used were: comparison of evolution of carbon dioxide with and without benzaldehyde and production of phenylacetylcarbinol after a single addition of benzaldehyde (0.2%) and after four additions (total of 0.8%). The highest decarboxylase activity in the presence of benzaldehyde was found withHansenula anomala, Saccharomyces carlsbergensis andSaccharomyces cerevisiae. InHansenula anomala, benzaldehyde caused a 16% inhibition of fermentation, in the other cultures inhibition lay between 35.5 and 63.2%. After a single addition of benzaldehyde the greatest amount of phenylacetylcarbinol was formed byHansenula anomala, Saccharomyces carlsbergensis andSaccharomyces cerevisiae. The yield of phenylacetylcarbinol in these cases was between 46.0% and 51.5 weight% (calculated on added benzaldehyde). During fementation with a higher benzaldehyde concentration,Saccharomyces carlsbergensis utilized 70% aldehyde for the formation of phenylacetylcarbinol while the rest was mostly reduced to benzylalcohol. Phenylacetylcarbinol was estimated after extraction of the fermentation medium with ether polarographically and polarimetrically, benzaldehyde was estimated polarographically directly in the medium. 相似文献
80.
Renata Miltko Grzegorz Bełżecki Andrzej Herman Barbara Kowalik Jacek Skomiał 《Archives of animal nutrition》2016,70(6):425-440
The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 104 (D. affine) to 42.2 · 104 cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 105 cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population. 相似文献