神经元保护蛋白TAT-Bcl-XL的体外高效表达及其功能的初步研究

目的：在原核表达系统中表达对凋亡神经元具有保护作用的重要蛋白Bcl-XL与蛋白质转导序列（PTD）的融合蛋白，并检测重组蛋白对重金属离子所诱导的细胞凋亡的保护作用。方法：通过RT-PCR的方法，用Bcl-XL特异引物从乳腺癌细胞系MCF-7细胞的总RNA中扩增出Bcl-XL基因，构建相应的原核表达载体，体外表达的TAT-Bcl-XL融合蛋白经镍亲和层析介质纯化后，通过免疫荧光方法检测其转导293T细胞的能力；并用流式细胞仪检测融合蛋白抑制细胞凋亡的能力。结果：经RT-PCR从MCF-7细胞总RNA中得到相应的TAT-Bcl-XL基因片段，并将其克隆入pCRT7/CT-TOPO载体中，重组质粒pTBTOPO转化大肠杆菌后，在SDS-PAGE和Western blot的结果中出现了与预期分子量相同的蛋白条带和阳性信号，纯化的TAT-Bcl-XL重组蛋白经免疫荧光检测，主要分布于细胞的细胞质中。流式细胞仪的检测结果显示融合表达蛋白可以有效地抑制Zn2＋离子所诱导的细胞凋亡，使细胞的存活率提高40%。结论：TAT-Bcl-XL融合蛋白在大肠杆菌中获得高效表达，初步的功能性检测表明融合蛋白具有抑制细胞凋亡的功能。     

Modulation of the inhibitory action of ATP on acetylcholine-activated current by protein phosphorylation in rat sympathetic neurons

Modulation by protein phosphorylation of the relation between acetylcholine (ACh)-activated current (I _ACh) and adenosine triphosphate-(ATP)-activated current (I _ATP) was investigated with the whole-cell voltage-clamp technique in rat sympathetic neurons. During simultaneous activation by 100 M ATP of an inward current, the current evoked by 100 M ACh was reduced to 60–70% of that in the absence of ATP. Effects of compounds that are known to modulate protein phosphorylation were tested by including them in the intracellular solution. The reduction ofI _ACh by ATP was not observed when K252a (1 M), a non-selective protein kinase inhibitor, adenosine 5-O-(3-thiotriphosphate) (ATP[S], 1 mM) or,-methylene ATP (1 mM) were included in the intracellular solution. Activators of protein kinases, adenosine 3,5-cyclic monophosphate (cAMP, 100 M), guanosine 3,5-cyclic monophosphate (cGMP, 100 M), phorbol 12-myristate 13-acetate (PMA, 1 M), also abolished the reduction by ATP ofI _ACh. The effects of okadaic acid, a protein phosphatase inhibitor, were paradoxical: okadaic acid (2 M) itself abolished the reduction by ATP ofI _ACh but it antagonized the abolishment by cAMP or cGMP of the reduction ofI _ACh. Okadaic acid did not affect the disappearance of the reduction ofI _ACh by ATP in the presence of intracellular PMA. The results suggest that the interaction betweenI _ACh andI _ATP is regulated by protein phosphorylation/dephosphorylation. Possible mechanisms underlying the effects of these modulators of protein phosphorylation are discussed.     

Isolation,characterization, phosphorylation and site of synthesis of Spinacia chloroplast ribosomal proteins

Summary We have characterized the ribosomal proteins from Spinacia chloroplasts using two-dimensional gel electrophoresis. The 30S and 50S subunits contain 23–25 and 36 ribosomal proteins, respectively. In contrast to prokaryotic ribosomes, chloroplast ribosomes contain at least one (and possibly two) phosphorylated ribosomal proteins. Isolated chloroplasts synthesize in the presence of (³⁵S) labeled methionine and cysteine at least seven 30S and thirteen 50S ribosomal proteins which are assembled into (pre)ribosomes. This suggests that about one third of the chloroplast ribosomal proteins is encoded by the chloroplast DNA itself. The identity of several labeled proteins in the two-dimensional gel electrophoretic patterns which did not comigrate with stained chloroplast ribosomal proteins is discussed.Abbreviations CBB Coomassie Brilliant Blue - CHI cycloheximide - cp chloroplast - DTT dithiotreitol - EDTA ethylene diamine tetraacetate - EGTA ethylene glycol-bis (-amino ethyl ether) N,N-tetraacetic acid - kD kilodalton - LHCP light harvesting chlorophyll a/b protein - PMSF phenyl methyl sulfonyl fluoride - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodiumdodecylsulphate     

Elucidation of the protein kinase C-dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL-lpr/lpr mice

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-+/+ mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4⁺ T lymphocytes and CD8⁺ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8⁺ T lymphocytes, but not CD4⁺ T lymphocytes, from MRL-+/+ mice were susceptible to the reagent. Interestingly, B220⁺Thy-1⁺CD4^?CD8^? T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-α level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-+/+. These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4⁺CD8⁺ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-+/+ mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4⁺ CD8⁺ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.     

In Situ Thermal Denaturation of Proteins in Dunning AT-1 Prostate Cancer Cells: Implication for Hyperthermic Cell Injury

The in situ thermal protein denaturation and its correlation with direct hyperthermic cell injury in Dunning AT-1 prostate tumor cells were investigated in this study. The in situ thermal protein denaturation was studied using both Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The FTIR spectra at different temperatures show changes in protein secondary structure (from alpha helix to extended beta sheet) during in situ thermal protein denaturation within AT-1 cells. Calorimetric studies using DSC show that endothermic heat release is associated with the in situ thermal protein denaturation. Furthermore, both the secondary structure changes detected by FTIR and the calorimetric changes detected by DSC were quantified and the kinetics of the overall in situ thermal protein denaturation was derived under different heating conditions. The onset temperature where the overall in situ thermal protein denaturation is first detectable was found to be scanning rate dependent (approximately 41 degrees C at 2 degrees C min(-1) and approximately 44 degrees C at 5 degrees C min(-1)). The kinetics of the overall in situ thermal protein denaturation was derived from both DSC and FTIR measurements and was fit using kinetic and statistical models. The kinetic data determined by FTIR and DSC under the same heating conditions match well with each other. The activation energy of the overall in situ thermal protein denaturation is found to be strongly dependent on the temperature range considered (the activation energy ranges from approximately 110 kJ mol(-1) between 44 and 90 degrees C to approximately 750 kJ mol(-1) between 44 and 50 degrees C). However, its dependence on heating rate is negligible. Several denaturation peaks, including a dominant one between approximately 62 and 65 degrees C, are identifiable from both the DSC and the FTIR results. To investigate directly the relationship between thermally induced cell injury and the in situ thermal protein denaturation, both acute (propidium iodide dye exclusion, assessed 3-h postthermal treatment) and chronic (clonogenics, assessed 7-day postthermal treatment) cell injury were quantified using AT-1 cells prepared under the same conditions as for the DSC protein studies. Comparisons of the results from the cell injury studies and the DSC protein denaturation studies show that the overall in situ thermal protein denaturation correlates well with both the acute and the chronic cell injury, which suggests that overall in situ thermal protein denaturation is an important mechanism of direct hyperthermic cell injury in AT-1 cells at the macromolecular level.     

Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities

 Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop. In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow, we stably transfected LLC-PK₁ and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK₁ clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane, including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl₂. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds. Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997     

Presenilin regulates extracellular regulated kinase (Erk) activity by a protein kinase C alpha dependent mechanism

Presenilin (PS1 and PS2) mutations cause early-onset familial Alzheimer's disease (AD). In addition to affecting β-amyloid precursor protein (APP) processing and Aβ generation, PSs regulate a number of signaling pathways. We previously showed that PSs regulate both phospholipase C (PLC) and protein kinase C (PKC) α and γ activities. We also reported that PS double knockout mouse embryonic fibroblasts (MEFs) have reduced levels of PKCα and enhanced levels of PKCδ. Here, we determined whether the PS modulation of PLC/PKC has consequences for extracellular regulated kinase (Erk) signaling. Erk has been suggested to be important in AD pathology by modulating APP processing and tau phosphorylation. We found that knocking out PS1 or PS2 alone resulted in increased Erk activity and that this effect could be reversed by the PKCα inhibitor Gö6976. We also found that Erk activity following either PLC or PKC stimulation was significantly lower in PS double knockout cells and that treatment with the PKC activator phorbol 12,13-dibutyrate (PdBu) down-regulated total-Erk levels in all cells except PS double knockouts. These results demonstrate that PSs regulate Erk activity through a PKCα dependent pathway and that disruption of PLC/PKC signaling in the absence of both PS1 and PS2 results in lower downstream activation of Erk.     

Influence of dietary protein level on protein self-selection and plasma and brain amino acid concentrations

Control of protein intake was studied in young rats that were allowed to choose between either protein-free and 55% casein diets or 15% and 55% casein diets. Animals on the protein-free vs. 55% casein regimen exhibited a lower weight gain, a lower cumulative energy intake and a greater cumulative total protein intake during the 13-day study compared to rats selecting between 15% and 55% casein. The daily average proportion of total food selected as casein by animals choosing between protein-free and 55% casein diets increased from 15% to 38% during the course of the study. In contrast, rats choosing between 15% and 55% casein chose 18-22% of total food as protein throughout the entire study. Long-term protein intake or protein selection did not correlate significantly with whole-brain contents of 5-HT or 5-HIAA. Our results suggest that protein intake is not regulated at a constant proportion of total calories, but is controlled between a minimum level that will support rapid growth and a maximum that, if exceeded, would require the animal to undergo substantial metabolic adaptation. The mechanism controlling protein selection may involve diet-induced changes in the brain content of total free indispensable amino acids.     

Postnatal expression of transport proteins involved in acid–base transport in mouse kidney

The kidney plays a major role in maintaining and controlling systemic acid–base homeostasis by reabsorbing bicarbonate and secreting protons and acid-equivalents, respectively. During postnatal kidney development and adaptation to changing diets, plasma bicarbonate levels are increasing, the capacity for urinary acidification maturates, and the final morphology and distribution of intercalated cells is achieved. In adult kidney, at least two types of intercalated cells (IC) are found along the collecting duct characterised either by the expression of AE1 (type A IC) or pendrin (non-type A IC) where non-type A IC are found only in the convoluted distal tubule, connecting tubule and cortical collecting duct. Here we investigated in mouse kidney the relative mRNA abundance, protein expression levels and distribution of several proteins involved in renal acid–base transport, namely, the Na⁺/HCO₃ ^– cotransporter NBC1 (SLC4A4), the Na⁺/H⁺-exchanger NHE3 (SLC9A3), two subunits of the vacuolar H⁺-ATPase [ATP6V0A4 (a4), ATP6V1B1 (B1)], the Cl^–/HCO₃ ^– exchangers AE1 (SLC4A1) and pendrin (SLC26A4). Relative mRNA abundance of all transport proteins was lowest at day 3 after birth and increased thereafter in parallel with protein levels. The numbers of type A and non-type A IC in the cortical collecting duct (CCD) increased from day 3 to days 18 and 24, whereas the number of IC in the CCD with apical staining for the vacuolar H⁺-ATPase subunits a4 and B1 decreased from day 3 to days 18 and 24, respectively. In addition, cells with characteristics of non-type A IC (pendrin expression, basolateral expression of vacuolar H⁺-ATPase subunits) were found in the inner and outer medulla 3 days after birth but were absent from the medulla of 24-day-old mice. Taken together, these results demonstrate massive changes in mRNA and protein expression levels of several acid–base transporters during postnatal kidney maturation and also show changes in intercalated cell phenotype in the medulla during these processes.     

mic2/CD99在经典型霍奇金淋巴瘤H/RS细胞中的表达及与Eber-1/LMP-1相关性的研究

目的： 检测mic2基因与CD99蛋白和Eber-1基因与潜伏膜蛋白-1（LMP1）在经典型霍奇金淋巴瘤（cHL）H/RS细胞中的表达，探讨mic2/CD99表达与Eber-1/LMP-1的相关性。 方法： 采用分子原位杂交和免疫组化技术并结合组织芯片技术检测59例石蜡包埋淋巴瘤组织标本［43例cHL,16例非霍奇金淋巴瘤（NHL）］mic2/CD99和Eber-1/LMP-1的表达，比较分析mic2/CD99在两组中的表达及与Eber-1/LMP-1的关系。 结果： cHL组CD99蛋白表达阳性率为2.3％，mic2基因表达为55.8％，LMP1表达为58.1％，Eber-1表达为53.5％；NHL组CD99蛋白与mic2基因表达高于cHL组（P<0.05）；LMP1和Eber-1的表达低于cHL组（P<0.05）；mic2基因的表达高于CD99蛋白的表达（P<0.05）；Eber-1与LMP1的表达无统计学意义（P>0.05）。CD99蛋白与LMP1的表达呈负相关（P<0.05），各项指标的表达与性别无关。CD99蛋白与LMP1的表达与年龄呈负相关（P<0.05），mic2与Eber-1的表达与年龄无关（P>0.05）。 结论： CD99蛋白在cHL H/RS细胞中低表达，并与LMP1的表达呈负相关。