血浆置换治疗慢性重型肝炎影响疗效的因素分析

目的分析血浆置换治疗慢性重型肝炎影响疗效的因素。方法回顾性分析血浆置换治疗慢性重型肝炎86例的临床资料,对影响临床疗效的因素进行分析。结果49例患者痊愈或明显好转,存活率为56.98%;37例病情恶化或死亡,病死率43.02%。死亡组年龄大于存活组(P<0.01);在肝硬化基础上发生的重型肝炎患者血浆置换的疗效低于在慢性肝炎基础上发生的重型肝炎(P<0.01);存活组凝血酶原活动度(PTA)、血清总胆固醇、甲胎蛋白及白蛋白水平高于死亡组,而血清总胆红素、内毒素水平低于死亡组(P<0.01);血浆置换治疗慢性重型肝炎早、中期患者疗效高于晚期患者疗效(P<0.01),慢性重型肝炎存在的并发症也影响血浆置换的疗效。讨论血浆置换的疗效与患者的年龄、治疗前的血清总胆红素、凝血酶原活动度(PTA)、血清总胆固醇、甲胎蛋白、白蛋白及内毒素水平有关。血浆置换治疗后胆红素水平反跳明显、PTA恢复不明显或甚至更低者的疗效差。我们认为血浆置换治疗>5或6次后病情仍未改善,血清胆红素水平下降不明显或反跳幅度明显、PTA恢复不明显或甚至更低者,有条件者应尽早施行肝移植术。     

Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells

BACKGROUND & AIMS: Increased serotonin levels have been implicated in the pathophysiology of diarrhea associated with celiac and inflammatory diseases. However, the effects of serotonin on Na+ /H+ exchange (NHE) activity in the human intestine have not been investigated fully. The present studies examined the acute effects of 5-hydroxytryptamine (5-HT) on NHE activity using Caco-2 cells as an in vitro model. METHODS: Caco-2 cells were treated with 5-HT (.1 micromol/L, 1 h) and NHE activity was measured as ethyl-isopropyl-amiloride (EIPA)-sensitive 22Na uptake. The effect of 5-HT receptor-specific agonists and antagonists was examined. The role of signaling intermediates in 5-HT-mediated effects on NHE activity was elucidated using pharmacologic inhibitors and immunoblotting. RESULTS: NHE activity was inhibited significantly (approximately 50%-75%, P < .05) by .1 micromol/L 5-HT via inhibition of maximal velocity (Vmax) without any changes in apparent affinity (Km) for the substrate Na+ . NHE inhibition involved a decrease of both NHE2 and NHE3 activities. Studies using specific inhibitors and agonists showed that the effects of 5-HT were mediated by 5-HT4 receptors. 5-HT-mediated inhibition of NHE activity was dependent on phosphorylation of phospholipase C gamma 1 (PLC gamma 1) via activation of src-kinases. Signaling pathways downstream of PLC gamma 1 involved increase of intracellular Ca 2+ levels and subsequent activation of protein kinase C alpha (PKC alpha). The effects of 5-HT on NHE activity were not cell-line specific because T84 cells also showed NHE inhibition. CONCLUSIONS: A better understanding of the regulation of Na+ absorption by 5-HT offers the potential for providing insights into molecular and cellular mechanisms involved in various diarrheal and inflammatory disorders.     

Mechanistic basis for ubiquitin modulation of a protein energy landscape

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other nondegradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen–deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Nevertheless, destabilization appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a nondestabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single posttranslational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality control properties.

Ubiquitin is an 8.5-kDa protein appended to target proteins as a posttranslational modification (PTM). Typically, ubiquitin is conjugated to the primary amine of substrate lysine residues, though noncanonical linkages to serine and cysteine also exist in vivo. Ubiquitin itself contains seven lysine residues, which allows building of ubiquitin chains with various linkages and topologies. Ubiquitination is most typically associated with targeting condemned proteins to the 26S proteasome for degradation; however, it is also involved in a large and ever-growing list of crucial regulatory, nondegradative cellular processes (1). A complex and highly regulated enzymatic cascade attaches ubiquitin to substrates and therefore plays a key role in determining the specific downstream effects of an individual ubiquitination event. There are several hundred E3 ligases, the terminal enzymes in this cascade (2), which give rise to broad proteome coverage and allow for some level of site specificity (3, 4).Multiple different ubiquitin chain linkages and topologies bind with high affinity to proteasomal ubiquitin receptors and promote degradation (5–8). However, the presence of a ubiquitin tag alone is not sufficient to ensure proteasomal degradation. In fact, a substantial proportion of ubiquitin-modified proteins that interact with the 26S proteasome are ultimately released (9, 10) and not degraded. The proteasome also relies on substrate conformational properties, initiating degradation at an unstructured region on the condemned protein (11, 12). Much work has been done to understand the requirements of this unstructured region with regard to length, sequence composition, and topological position (13–15), yet at least 30% of known proteasome clients lack such a region (16). While evidence suggests that well-folded proteins are processed by diverse cellular unfoldases, such as Cdc48/p97/VCP (17, 18), an intriguing possibility is that the ubiquitin modification itself can modulate the conformational landscape and thus regulate proteasome substrate selection. Simulations have suggested that ubiquitination can destabilize the folded state of the substrate protein, thereby allowing it to more readily adopt unfolded or partially unfolded conformations (19, 20).Recently, we demonstrated that this is indeed the case: ubiquitin can exert significant effects on a substrate’s energy landscape depending on the site of ubiquitination and the identity of the substrate protein. Moreover, these changes can have direct consequences for proteasomal processing (21). By examining the energetic effects of native, isopeptide-linked ubiquitin attachment to three different sites within the small protein barstar from Bacillus amyloliquefaciens, we found that ubiquitin attached at either lysine 2 or lysine 60 destabilizes the protein both globally and via subglobal fluctuations, and we thus refer to these residues as sensitive sites. By contrast, ubiquitination at lysine 78 produces little effect on the energy landscape (21), and we therefore term it a nonsensitive site. Another study found that ubiquitin, appended through a nonnative linkage, can destabilize a folded substrate as measured by changes in the midpoints for thermally induced unfolding transitions (22).Ubiquitination at the two sensitive sites in barstar increases the population of partially unfolded, high-energy states on the landscape sufficient for proteasomal engagement and degradation. Ubiquitination at the single nondestabilizing site does not allow for proteasomal degradation. These results suggest that ubiquitin-mediated destabilization can reveal an obligate unstructured region in substrates that otherwise lack such a region. Furthermore, ubiquitination at sensitive sites results in more rapid degradation of these barstar variants when a proteasome-engageable unstructured tail is fused to their C termini (21).This previous work clearly demonstrates that ubiquitin-mediated changes to the protein landscape can play an important role in proteasomal selectivity and processing; it did not, however, uncover the molecular mechanisms through which these site-specific effects arise. Here, we interrogate the molecular mechanisms of ubiquitin-induced changes for these same single-lysine variants of barstar. We investigate differences in the intrinsic dynamics of these regions within barstar and differences in how the protein responds to ubiquitination at these individual sites. We employed two sets of complementary approaches: 1) NMR and HDX-MS (hydrogen–deuterium exchange mass spectrometry) to characterize the equilibrium conformational fluctuations of the substrate protein in the presence and absence of ubiquitin and 2) molecular dynamics (MD) simulations to track the position of every atom in barstar, in the presence and absence of ubiquitin, starting from its native conformation over the timescale of microseconds.We find that ubiquitination has only subtle effects on the native structure of barstar. Ubiquitination at the sensitive sites, however, selectively increases fluctuations that expose barstar’s C terminus. While both of the sensitive sites arise in regions of barstar with relatively high conformational flexibility, the observed destabilization appears to occur through different thermodynamic mechanisms. By contrast, ubiquitination at the nonsensitive site has a protective effect on barstar’s C terminus. Thus, the effects of ubiquitination at each site are highly dependent on the local context. This mechanistic understanding of the site-specific effects of ubiquitination should aid in developing predictive models of the energetic consequences of individual ubiquitination events and also of the ways in which aberrant lysine targeting leads to disease (23–25).     

Two Patients with Acute Guillain-Barré Syndrome Treated with Different Apheresis Methods

Abstract: We treated 2 patients with severe acute Guillain-Barré syndrome (GBS) with different apheresis methods. Sera from these patients in the acute phase contained a high titer of immunoglobulin (1g)G anti-GM1 ganglioside antibody. One was a 57-year-old female treated with immunoadsorption (IA). IgG anti-GM1 antibody titer was reduced from 1:102,400 to 1:6,400 measured by ELISA after 1 month. However, her residural deficit was severe. The other patient was a 43-year-old male treated with plasma exchange (PE). IgG anti-GM1 antibody was reduced from 1:3,200 to 1:400 after 1 session of PE. After 1 month, he could walk independently. The better prognosis of the latter was possibly because of the patient's younger age and the plasma exchange that might have reduced such pathogenic factors as antiganglioside antibodies at the early stage of GBS.—     

Sodium-Hydrogen Exchange and Platelet Function

On stimulation of platelets with agonists, for example, thrombin, a rapid rise in intracellular pH is observed. This alkalinization is mediated by an increase in transport activity of the Na⁺/H⁺ exchanger isoform NHE1. In addition to this Na⁺/H⁺ exchange mechanism, platelets express bicarbonate/chloride exchangers, which also contribute to pH_i homeostasis. The main functions of NHE1 in platelets include pH_i control, volume regulation, and participation in cell signaling. The isoform NHE1 is highly sensitive toward inhibition by EIPA, Hoe694, and Hoe642. The regulation of NHE1 activity is complex and is not completely understood. It includes the MAP kinase cascade, the Ca/calmodulin system, several heterotrimeric G proteins (G12, G13, Gq, and Gi), small G proteins (ras, cdc42, rhoA), and downstream kinases (e.g., p160ROCK). Volume challenges stimulate tyrosine phosphorylation of cytoplasmic proteins, which ultimately activate NHE1. Thrombin, thromboxane, platelet-activating factor, angiotensin II, endothelin, phorbol ester, and Ca²⁺ ionophors stimulate NHE1 activity in platelets. Blockade of platelet NHE1 can inhibit platelet activation. With the development of highly specific NHE1 inhibitors, detailed investigation of the relationships between NHE1 activity and platelet activation now becomes feasible.     

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at lambda >495 nm. Subsequent irradiation of the complex at lambda310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with T(alpha). For identification of the sites of crosslinks in T(alpha), the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the T(alpha) species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting T(alpha) derivatives and isolation of T(alpha) peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in T(alpha) containing the amino acid sequences 310-313 and 342-345.     

The metabolites of nitric oxide in sickle-cell disease

Summary. Plasma NOx concentrations were raised in 22 acute painful crises in SCD. We have measured blood concentrations of nitric oxide metabolites (NOx) in sickle-cell disease (SCD), and shown that they are increased compared with healthy controls (P = 0.002), and haemoglobin E/β-thalassaemic controls (P=0.05). Concentrations in steady-state SCD were also higher than in healthy controls (P = 0.04) but not significantly different from the concentrations at the beginning of painful crises (P = 0.34). Importantly, in 12 regularly exchanged sicklers, the mean pre-transfusion NOx concentration did not differ significantly from the control population (P=0.52), suggesting that the changes in NO metabolism can be reversed. It is unlikely that the increased concentrations of NOx in SCD result from anaemia or haemolysis as the untransfused haemoglobin E/β-thalassaemics did not show increased levels.     

地方性氟中毒患者外周血淋巴细胞姐妹染色单体交换的研究

用荧光加 Giemsa 技术,区分姐妹染色单体,分析92人外周血淋巴细胞的 SCE,比较地方性氟中毒患者和对照组 SCE 频率变化,发现53名氟中毒患者 SCE 频率(9.15±2.39)高于39名对照组(4.56±0.88),P<0.01。认为 SCE 增高是由高氟引起,氟是一种诱变因素。SCE频率的增高是高氟造成遗传物质损伤的证据之一。     

Laboratory abnormalities in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura is an uncommon disorder that requires prompt recognition and intervention to prevent death. To date, information regarding the classic laboratory abnormalities in the disease has been derived from small numbers of patients whose laboratory tests have been done at many different sites. We report the laboratory findings in 135 patients who presented with thrombotic thrombocytopenic purpura to 17 Canadian centres. 50 men and 85 women had a mean platelet count of 25.3 ± 19.4 × 10⁹/l. The initial platelet count correlated with mortality; 32% of patients with a platelet count of 20 × 10⁹/l or less died compared with 18% of patients with a platelet count >20 × 10⁹/l ( P  = 0.058). The platelet-associated IgG was elevated in 88% at presentation whereas the indirect platelet suspension immunofluorescence test was positive in only 18%. 93% of the sera showed reactivity against platelets following protein blotting. All sera tested also showed reactivity against endothelial cells. Immune complexes were seen in all patients, whereas the platelet aggregating factor was detected in 59%. Although the von Willebrand factor was elevated in the majority of patients at entry, the multimer pattern was variable and showed no predictive pattern. Renal dysfunction was common (18%).     

β2-Agonists and running economy in prepubertal boys

Asthmatic athletes (adults and junior) have competed successfully at the highest level for many years assisted by pre-event medication with β2-agonists. To examine the impact of (β2–agonists upon submaximal running economy (oxygen consumption at a given submaximal work load), we studied 10 nonasthmatic boys (age, 10.4 ± 0.48 years, mean ± SD). They each completed submaximal (speeds, 7.2, 8.0, and 8.8 km/hr) and peak treadmill running protocols preceded by treatment with β2-agonist (terbutaline, 500 (μg via nebuhaler) or placebo in a randomized, crossover single-blind study. No significant differences were found between running economy and heart rate during the submaximal exercise tests or between peak oxygen consumption (V₀₂), peak respiratory exchange ratio, peak heart rate (HR), or total running time during the peak V₀₂ test. Pretreatment with terbutaline did produce small but nonsignificant increases in aerobic fractional utilization (percent peak V₀₂ on drug: 65.9%, 72.6%, and 76.7% vs. placebo: 65.1%, 70%, and 75.5%), at the three submaximal work loads. Respiratory exchange ratio (RER) values were elevated throughout the submaximal tests (on drug: 0.94, 0.93, and 0.94 vs. placebo: 0.91, 0.92, and 0.91, P < 0.05). No significant differences were found between drug and placebo for minute ventilation (V_E) and ventilatory equivalent for oxygen (V_E/V₀₂), at both submaximal and peak exercise intensities. The significant increase in forced expiratory volume in 1 second (FEV₁) (% predicted) found following submaximal (pre: 107 ± 13 vs. post: 111 ± 17, P < 0.001) and peak exercise (pre: 103 ± 10 vs. post: 106 ± 14, P < 0.001), both with drug and placebo, suggests an effect of exercise rather than of drug. These results indicate that therapeutic doses of an inhaled β2–agonist administered immediately pre-exercise do not enhance the submaximal running economy or peak V₀₂ of nonasthmatic children.