Wortmannin-mediated inhibition of phosphatidylinositol 3-kinase activity triggers apoptosis in normal and neoplastic B lymphocytes which are in cell cycle

The B cell functional response following ligation of surface(s) lgM is dependent upon the differentiation stage of the populationstudied: cross-linking slgM promotes proliferation of restingtonsillar follicular mantle (FM) B lymphocytes but induces apoptosisin the susceptible Epstein- Barr virus genome-negative Burkittlymphoma (BL) cell line Ramos (Ramos-BL). This study investigateswhether phosphatidylinositol-3-kinase (Pl3-kinase), which hasbeen reported to be intimately involved in the regulation ofcellular growth, plays a role in the regulation of these sig-promoted B cell responses, and uses the selective and irreversibleinhibitor of Pl3-kinase activity, wortmannin (Wm). In Ramos-BLB cells, at 8 h post-treatment, Wm triggers a transient increasein apoptosis of 16 ± 6.9% with a concomitant cellularloss of 16 ± 6.1% from the G₁ phase of cell cycle; [³H]thymidineincorporation also decreases by 33 ± 5.0%, from 37,274c.p.m. ± 10% to 25,127 c.p.m. ± 4.0%. Moreover,at 72 h culture, Wm inhibits anti-lgM-induced FM B lymphocytelevels of [³H]thymidine incorporation typically by 47% and triggers80% apoptosis from the G₀G₁ phase of cell cycle. Ramos-BL Bcells exhibit high basal levels of Pl3-kinase activity, as determinedby immunoprecipitation with antibody to the p85 regulatory subunitof Pl3-kinase and ³²P incorporation into phosphatidylinositol,which is not significantly affected by anti-lgM stimulation;by contrast, anti-lgM stimulates significant Pl3-kinase activityover negligible basal levels in FM B lymphocytes. Pre-treatmentwith Wm inhibits Pl3-kinase activity in both cell types. Takentogether these data indicate that in Ramos-BL B cells slgM-triggeredgrowth arrest and apoptosis is Pl3- kinase independent, whereasPl3-kinase activity is critical for slgM-triggered mitogenesisof FM B lymphocytes. Thus Pl3-kinase plays a pivotal role inthe regulation of both normal and neoplastic B lymphocyte progressionthrough the cell cycle, such that if this Pl3-kinase-dependentpathway is inhibited these cells default to apoptosis.     

REPERFUSION FOLLOWING MYOCARDIAL ISCHAEMIA ENHANCES INOSITOL PHOSPHATE RELEASE IN THE ISOLATED PERFUSED RAT HEART

1. Global myocariial ischaemia (MI) for periods greater tan 5 min caused an inhibition of phosphatidylinositol specific phospholipase C (PtdIns-PLC) activity. 2. Two min reperfusion following a 20 min MI period, a time point associated with reperfusion-induced arrhythmias, resulted in an activation of PtdIns-PLC activity, dependent on endogenous noradrenaline and mediated via al-adrenoceptors. 3. This 2 min reperfusion response, in contrast to healthy myocardium, resulted in: (i) enhanced PtdIns-PLC activity; (ii) increased sensitivity to endogenous noradrenaline; (iii) rapid increases in inositol(1,4,5)trisphosphate (Ins(1,4,5)P3); and (iv) PLC hydrolysis primarily of PtdIns(4,5)P₂, such that the majority of InsP isomers derive from Ins(1,4,5)P₃. 4. Together, these data suggest a functional role for Ins(1,4,5)P3 under postischaemic reperfusion conditions, and provide a possible link between al-adrenoceptor stimulation of the PtdIns turnover pathway and reperfusion injury.     

25种常用食物总脂质、总磷脂和主要磷脂含量的比较

<正> 磷脂是构成生物细胞膜的基本组分,以其规律的结构保证细胞的正常形态和功能。因而经常有一定质量的磷脂保证,是维持所有细胞健康生长和生理功能的基础。磷脂已被广泛应用为食品添加剂,由于磷脂是一种天然产物,更加适应当今世界对“非化学添加物”的需要。但目前的食物成分表均无磷脂的含量,为此对云南、陕西、辽宁等地常用食物中总脂质、总磷脂和主要磷脂的含量进行了分析。     

Transient Brain Ischaemia Provokes Ca2+, PIP2 and Calpain Responses Prior to Delayed Neuronal Death in Monkeys

To clarify the mechanism of postischaemic delayed cornu Ammonis (CA)-1 neuronal death, we studied correlations among calpain activation and its subcellular localization, the immunoreactivity of phosphatidylinositol 4,5-bisphosphate (PIP₂) and Ca²⁺ mobilization in the monkey hippocampus by two independent experimental approaches: in vivo transient brain ischaemia and in vitro hypoxia-hypoglycaemia of hippocampal acute slices. The CA-1 sector undergoing 20 min of ischaemia in vivo showed microscopically a small number of neuronal deaths on day 1 and almost global neuronal loss on day 5 after ischaemia. Immediately after ischaemia, CA-1 neurons ultrastructurally showed vacuolation and/or disruption of the lysosomes. Western blotting using antibodies against inactivated or activated μ-calpain demonstrated μ-calpain activation specifically in the CA-1 sector immediately after ischaemia. This finding was confirmed in the perikarya of CA-1 neurons by immunohistochemistry. CA-1 neurons on day 1 showed sustained activation of μ-calpain, and increased immunostaining for inactivated and activated forms of μ- and m-calpains and for PIP₂. Activated μ-calpain and PIP₂ were found to be localized at the vacuolated lysosomal membrane or endoplasmic reticulum and mitochondrial membrane respectively, by immunoelectron microscopy. Calcium imaging data using hippocampal acute slices showed that hypoxia-hypoglycaemia in vitro provoked intense Ca²⁺ mobilization with increased PIP₂ immunostaining specifically in CA-1 neurons. These data suggest that transient brain ischaemia increases intracellular Ca²⁺ and PIP₂ breakdown, which will activate calpain proteolytic activity. Therefore, we suggest that activated calpain at the lysosomal membrane, with the possible release of biodegrading enzyme, will cause postischaemic CA-1 neuronal death.     

Arachidonic acid modulates [14C]stearic acid incorporation into phosphatidylinositol,in human neuroblastoma cells

In the human neuroblastoma cell line SK-N-BE(2), arachidonic acid (AA), supplied in the medium at micromolar concentrations, markedly ehnanced [¹⁴C]stearic acid (SA) (but not [¹⁴C]palmitic acid or [¹⁴C]oleic acid) incorporation into phosphatidylinositol (PtdIns). AA failed to stimulate [¹⁴C]SA incorporation into PtdIns precursors, namely phosphatidic acid and cytidinediphosphodiacylglycerol; furthermore, enhanced [¹⁴C]SA incorporation, brought about by exogenously administered AA, was not restricted to PtdIns tetraenoic species. When cells were pulsed for 1 h with [¹⁴C]SA (either in the presence or absence of AA) and then reincubated in AA- and [¹⁴C]SA-free medium, a marked loss of radioactivity from PtdIns was observed, that however was restricted to molecular species other than tetraenoic. These results are discussed in the light of possible mechanisms through which PtdIns achieves the 1-stearoyl-2-arachidonoyl configuration.     

抑制生存传导路径可增加肿瘤细胞化疗敏感性

目的：研究抑制磷酸酰肌醇3激酶(PI3K)／Akt生存传导路径是否可增加肿瘤细胞对一些化疗药物的敏感性。方法：采用Akt亚型特异抑制剂结合多柔比星(阿霉素ADM)或喜树碱治疗各种肿瘤细胞系；检测细胞凋亡蛋白激酶3活性，来定量评估细胞凋亡程度；免疫沉淀 Werstern印迹法测定药物对磷酸化各类Akt亚型的抑制作用。结果：①各类Akt亚型抑制剂可阻断Akt1或Akt2蛋白序列上的第308位点苏氨酸(pAkt^308)和第473位点丝氨酸(pAkt^473)磷酸化，并表现出相应的Akt亚型抑制特异性；②单独抑制某一类Akt亚型并不足以增加肿瘤细胞对化疗药物的敏感性，只有同时抑制Akt1和Akt2才能增敏药物对肿瘤细胞的杀灭效应；③药物的增敏效应可能和肿瘤细胞内源性PTEN脱磷酸酶的表达有一定的关联。结论：临床应用化疗结合PI3K／Akt生存传导路径抑制剂应选择具有阻断2种Akt亚型功能的药物以达到最大的肿瘤杀灭效应。     

Glycosylphosphatidylinositol (GPI) anchored protein deficiency serves as a reliable reporter of Pig‐a gene Mutation: Support from an in vitro assay based on L5178Y/Tk+/− cells and the CD90.2 antigen

Lack of cell surface glycosylphosphatidylinositol (GPI)‐anchored protein(s) has been used as a reporter of Pig‐a gene mutation in several model systems. As an extension of this work, our laboratory initiated development of an in vitro mutation assay based on the flow cytometric assessment of CD90.2 expression on the cell surface of the mouse lymphoma cell line L5178Y/Tk^+/?. Cells were exposed to mutagenic and nonmutagenic compounds for 24 hr followed by washout and incubation for an additional 7 days. Following this mutant manifestation time, cells were labeled with fluorescent antibodies against CD90.2 and CD45 antigens. These reagents indicated the presence of GPI‐anchored proteins and general cell surface membrane receptor integrity, respectively. Instrument set‐up was aided by parallel processing of a GPI anchor‐deficient subclone. Results show that the mutagens reproducibly caused increased frequencies of mutant phenotype cells, while the nonmutagens did not. Further modifications to the method, including application of a viability dye and an isotype control for instrument set‐up, were investigated. As a means to verify that the GPI‐anchored protein‐negative phenotype reflects bona fide Pig‐a gene mutation, sequencing was performed on 38 CD90.2‐negative L5178Y/Tk^+/? clones derived from cultures treated with ethyl methanesulfonate. All clones were found to have mutation(s) within the Pig‐a gene. The continued investigation of L5178Y/Tk^+/? cells, CD90.2 labeling, and flow cytometric analysis as the basis of an in vitro mutation assay is clearly supported by this work. These data also provide evidence of the reliability of using GPI anchor‐deficiency as a valid reporter of Pig‐a gene mutation. Environ. Mol. Mutagen. 59:18–29, 2018. © 2017 Wiley Periodicals, Inc.     

Hypothalamic insulin and glucagon-like peptide-1 levels in an animal model of depression and their effect on corticotropin-releasing hormone promoter gene activity in a hypothalamic cell line

Background

In depression, excessive glucocorticoid action may cause maladaptive brain changes, including in the pathways controlling energy metabolism. Insulin and glucagon-like peptide-1 (GLP-1), besides regulation of glucose homeostasis, also possess neurotrophic properties. Current study was aimed at investigating the influence of prenatal stress (PS) on insulin, GLP-1 and their receptor (IR and GLP-1R) levels in the hypothalamus. GLP-1 and GLP-1R were assayed also in the hippocampus and frontal cortex – brain regions mainly affected in depression. The second objective was to determine the influence of exendin-4 and insulin on CRH promoter gene activity in in vitro conditions.

Targeting the PI3K/AKT/mTOR pathway in biliary tract cancers: A review of current evidences and future perspectives

Biliary tract cancers (BTCs) are a group of invasive neoplasms, with increasing incidence and dismal prognosis. In advanced disease, the standard of care is represented by first-line chemotherapy with cisplatin and gemcitabine. In subsequent lines, no clear recommendations are currently available, highlighting the need for novel therapeutic approaches.The PI3K/AKT/mTOR pathway is a core regulator of cell metabolism, growth and survival, and is involved in BTCs carcinogenesis and progression. Mutations, gene copy number alterations and aberrant protein phosphorylation of PI3K, AKT, mTOR and PTEN have been thoroughly described in BTCs and correlate with poor survival outcomes.Several pre-clinical evidences state the efficacy of PI3K/AKT/mTOR pathway inhibitors in BTCs, both in vitro and in vivo. In the clinical setting, initial studies with rapamycin analogs have shown interesting activity with an acceptable toxicity profile. Novel strategies evaluating AKT and PI3K inhibitors have risen serious safety concerns, pointing out the need for improved patient selection and increased target specificity for the clinical development of these agents, both alone and in combination with chemotherapy.This review extensively describes the role of the PI3K/AKT/mTOR pathway in BTCs and examines the rationale of its targeting in these tumors, with particular focus on clinical activity, toxicities and perspectives on further development of PI3K/AKT/mTOR pathway inhibitors.     

Clinical Applications of Granulocyte Colony Stimulating Factor (G-CSF)

Abstract

The production of neutrophil granulocytes is a complex and dynamic process during which a small number of limited stem cells give rise to lineage specific progenitors that proliferate in the bone marrow subsequently entering the blood as mature polymorphonuclear leukocytes. One hematopoietic growth factor which appears to specifically control the proliferation and terminal maturation of this specific myeloid lineage is granulocyte colony stimulating factor.