DIA-Based Proteomics Identifies IDH2 as a Targetable Regulator of Acquired Drug Resistance in Chronic Myeloid Leukemia

Drug resistance is a critical obstacle to effective treatment in patients with chronic myeloid leukemia. To understand the underlying resistance mechanisms in response to imatinib mesylate (IMA) and adriamycin (ADR), the parental K562 cells were treated with low doses of IMA or ADR for 2 months to generate derivative cells with mild, intermediate, and severe resistance to the drugs as defined by their increasing resistance index. PulseDIA-based (DIA [data-independent acquisition]) quantitative proteomics was then employed to reveal the proteome changes in these resistant cells. In total, 7082 proteins from 98,232 peptides were identified and quantified from the dataset using four DIA software tools including OpenSWATH, Spectronaut, DIA-NN, and EncyclopeDIA. Sirtuin signaling pathway was found to be significantly enriched in both ADR-resistant and IMA-resistant K562 cells. In particular, isocitrate dehydrogenase (NADP(+)) 2 was identified as a potential drug target correlated with the drug resistance phenotype, and its inhibition by the antagonist AGI-6780 reversed the acquired resistance in K562 cells to either ADR or IMA. Together, our study has implicated isocitrate dehydrogenase (NADP(+)) 2 as a potential target that can be therapeutically leveraged to alleviate the drug resistance in K562 cells when treated with IMA and ADR.     

Effect of Chronic Lithium Treatment on 5-Hydroxytryptamine Autoreceptors and Release of 5-[3H]Hydroxytryptamine from Rat Brain Cortical, Hippocampal, and Hypothalamic Slices

The effect of acute and chronic lithium treatments on 5-hydroxytryptamine (5-HT, serotonin) release and on its regulation by presynaptic 5-HT autoreceptors was studied in [3H]5-HT preloaded superfused rat brain slices. The [3H]5-HT overflow evoked by a 30-s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium-containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5-HT release. The K+-induced release observed in both control and chronically lithium-treated animals was Ca2+-dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5-HT overflow in the cortex and hypothalamus but not in hippocampus [corrected]. The Ca2+-independent overflow induced by fenfluramine was also decreased in cortical slices from lithium-treated animals. The sensitivity of the inhibitory 5-HT autoreceptors was assessed by the response to the 5-HT agonist 5-methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5-HT release induced by 5-methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5-HT sites may be responsible for the increase in K+-stimulated 5-HT overflow in brain slices of animals treated chronically with lithium.     

Biological responses to overload training in endurance sports

Five subjects undertook 10 days of twice daily interval training sessions on a treadmill followed by 5 days of active recovery. On days 1, 6, 11, and 16 the subjects were required to undertake a test of submaximal and maximal work capacity on a treadmill combined with a performance test consisting of a run to exhaustion with the treadmill set at 18 km.h-1 and 1% gradient. Also on these days a pre-exercise blood sample was collected and analysed for a range of haematological, biochemical and immunological parameters. The subjects experienced a significant fall in performance on day 11 which had returned to pretraining levels on day 16. Serum ferritin concentrations were depressed significantly from pretraining concentrations at the conclusion of the recovery period while the expression of lymphocyte activation antigens (CD25+ and HLA-DR+) was increased both after the training phase and the recovery phase. The number of CD56+ cells in the peripheral circulation was depressed at the conclusion of the recovery period. Several parameters previously reported to change in association with overload training failing to reflect the decrease in performance experienced by subjects in this study, suggesting that overtraining may best be diagnosed through a multifactorial approach to the recognition of symptoms. The most important factor to consider may be a decrease in the level of performance following a regeneration period. The magnitude of this decreased performance necessary for the diagnosis of overtraining and the nature of an "appropriate" regeneration period are, however, difficult to define and may vary depending upon the training background of the subjects and the nature of the preceding training. It may or may not be associated with biochemical, haematological, physiological and immunological indicators. Individual cases may present a different range of symptoms and diagnosis of overtraining should not be excluded based on the failure of blood parameters to demonstrate variation. However, blood parameters may be useful to identify possible aetiology in each separate case report of over-training. An outstanding factor to emerge from this study was the difficulty associated with an objective diagnosis of overtraining and this is a possible reason why there have been new accounts of overtraining research in the literature.     

Low incidence of bronchus-associated lymphoid tissue (BALT) in chronically inflamed human lungs

The relevance of bronchus-associated lymphoid tissue (BALT) in man is still under discussion. Animal experiments indicate that the development of BALT is dependent on microbial stimulation. Therefore, the incidence of BALT was investigated retrospectively in specimens removed during surgical procedures on patients with chronic pulmonary inflammation. All these patients had severe chronic bronchitis and bronchiectasis, but BALT was found in only 8%. In patients with BALT and a malignant tumor, occlusion of a bronchus with poststenotic pneumonia was always present and BALT was observed exclusively in areas peripheral to the occlusion. In man other compartments of the lung must be responsible for the immune function of BALT found in animals. Partly presented at the Congress of the Eur. Respir. Soc, 21.- 26.9.1991 in Brussels. (Abstract: Eur Respir J. 4, Suppl. 14:217p)     

The effect of dissolved oxygen on the growth of young-of-the-year winter flounder,Pseudopleuronectes americanus

Synopsis The effects of constant and diurnally fluctuating levels of dissolved oxygen on the growth of young-of-the-year winter flounder,Pseudopleuronectes americanus, were examined under controlled laboratory conditions. Fish were exposed for either 10 or 11 weeks to constant levels of 6.7 (high) and 2.2 (low) mg l^–1, and a diurnal fluctuation, ranging from 2.5 to 6.4 mg 0₂l^–1. Growth rates, calculated for both standard length and weight, for fish exposed to low and diurnally fluctuating levels were significantly reduced (p < 0.001) as compared to those for fish exposed to the high level. Growth rates of fish exposed to the high level were over twice those of fish held under low oxygen conditions. Under fluctuating conditions, fish grew at intermediate rates. Following these exposures, all fish were subsequently held at 7.2 mg Oz l^–1 for five weeks. Growth rates increased over two and a half times for fish previously exposed to the low oxygen level and were significantly (p < 0.001) higher than for the other two groups.     

Quantitative autoradiographic analysis of [I]-human CGRP binding sites in the dorsal horn of rat following chronic constriction injury or dorsal rhizotomy

Quantitative receptor autoradiography was used to examine the binding of [¹²⁵I]-human CGRP in the dorsal horn of the L4 spinal segment of rats with a chronic constriction injury (CCI) of the sciatic nerve or unilateral dorsal rhizotomies of spinal segments L1–L6. At the times selected for study, we found no change in the amount of CGRP binding in any areas examined following CCI. In contrast, our results showed a temporally related increase in the amount of CGRP binding in areas within laminae I–II and in lateral lamina V of the dorsal horn ipsilateral to the rhizotomies. These results indicate that CGRP binding sites are regulated, most likely, by changes in the release of CGRP. Further, our results suggest that the release of CGRP from primary afferent neurons is unchanged in animals with a CCI.     

Cytotoxic and noncytotoxic mechanisms involved in the in vitro anti-leukaemia effects of T cell clones established from a chronic myelogenous leukaemia patient during treatment in vivo with interferon α

Summary T cell clones derived from a chronic myelogenous leukaemia (CML) patient during interferon (IFN, Wellferon) biotherapy preferentially lysed autologous rather than allogeneic CML target cells in an apparently MHC-unrestricted fashion, but also lysed bone marrow cells from certain normal donors regardless of whether or not they shared HLA antigens with the patient. Although T cell clones inhibited both CML and normal bone marrow in the colony-forming assay, they blocked proliferation of CML cells more efficiently than bone marrow cells. This inhibitory effect was mediated at least in part by the tumour necrosis factor (TNF) and IFN secreted by the clones. Antisera to these cytokines partially prevented inhibition. Involvement of additional factors is also suggested in blocking CML cell proliferation because this was not 100% inhibited even by a combination of TNF and IFN. In addition, most clones failed strongly to block the proliferation of normal bone marrow cells, which were susceptible to inhibition by these cytokines.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

慢性蜜蜂麻痹病病毒核酸与蛋白质组分的研究

从自然感染的意大利麻痹病蜂(APis mellifera)头部,经二次差速离心与蔗糖梯度离心获得纯化的慢性蜜蜂麻痹病病毒(CBPV)。纯化的CBPV制备物感染正常蜜蜂,4天后出现典型的麻痹症状,接着死亡,平均死亡率分别为95％与100％。SDS-聚丙烯酰胺凝胶电泳分析,二次差速离心初步纯化的病毒制备物含有多条蛋白带,而蔗糖密度梯度纯化的病毒制备物仅含有单一的多肽带。5％、7.5％与10％SDS-聚丙烯酰胺凝胶电泳分析,均检测出一种病毒蛋白质,分子量大约为24,200道尔顿,而且不同凝胶浓度检测的蛋白质分子量相近。慢性蜜蜂麻痹病病毒核酸也用SDS-聚丙烯酰胺凝胶电泳分析,结果表明,凝胶中有5条带,对核酸酶敏感,证明该病毒含有5个单股RNA组分。对慢性蜜蜂麻痹病病毒的基因组结构进行了讨论。     

Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108-15 Cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (N_i) and stimulatory (N_s) components. Inactivation of N_i by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of N_s, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E₁, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through N_i resulting in the unimpeded expression of N_s activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which N_i regulates adenylate cyclase in this cell line.     

Chronic administration of an organophosphorus insecticide to rats alters cholinergic muscarinic receptors in the pancreas

Male rats were treated for 10 days with the organophosphorus insecticide, acetylcholinesterase inhibitor, O,O-diethyl S-[2-(ethylthio)ethyl]phosphorodithioate (disulfoton, 2 mg/kg/day by gavage). At the end of the treatment, binding of [³H]quinuclidinyl benzilate ([³H]QNB) to cholinergic muscarinic receptors and cholinesterase (ChE) activity were assayed in the pancreas. Functional activity of pancreatic muscarinic receptor was investigated by determining carbachol-stimulated secretion of α-amylase in vitro. ChE activity and [³H]QNB binding were significantly decreased in the pancreas from disulfoton-treated rats. The alteration of [³H]QNB binding was due to a decrease in muscarinic receptor density with no change in the affinity. Basal secretion of amylase from pancreas in vitro was not altered, but carbachol-stimulated secretion was decreased. The effect appeared to be specific since pancreozymin was able to induce the same amylase release from pancreases of control and treated rats. The results suggest that repeated exposures to sublethal doses of an organophosphorus insecticide lead to a biochemical and functional alteration of cholinergic muscarinic receptors in the pancreas.