Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae

Fusions of the glycolytic genes TPI1, PGK1, ENO1, PYK1, PDC1, and ADH1 with the lacZ reporter gene of Escherichia coli and a lacZ fusion construct of a 390-bp fragment from the promoter of the HXT7 gene were assayed for β-galactosidase activity. The glycolytic promoters were induced after addition of glucose to ethanol-grown cells, whereas the HXT7 promoter fragment showed a constitutive β-galactosidase expression on both carbon sources. The genes coding for the seven enzymes of lower glycolysis Tdh, Pgk, Gpm, Eno, Pyk, Pdc, and Adh were simultaneously put under the control of the same strong promoter, a truncated HXT7 promoter that is constitutively active on ethanol as well as on glucose medium. Genomic expression of the glycolytic genes under the control of this promoter, resulted in an at least 2-fold overexpression. The gene MSG5 was isolated, coding for a protein phosphatase normally involved in cell cycle regulation, as a factor that possibly influences the expression of the HXT7 gene. However, overexpression of MSG5 had no effect on the expression of the HXT7/lacZ fusion, whereas a deletion of this gene resulted in a decreased expression of β-galactosidase.     

Proteomics analysis of chinese hamster ovary cells undergoing apoptosis during prolonged cultivation

The degradation of environmental conditions, such as nutrient depletion and accumulation of toxic waste products over time, often lead to premature apoptotic cell death in mammalian cell cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. To our knowledge, the work presented here is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells. Flow cytometry analyses of various activated caspases demonstrated the onset of apoptosis in Chinese hamster ovary cells during prolonged cultivation was primarily through the intrinsic pathway. Differential in gel electrophoresis proteomic study comparing protein samples collected during cultivation resulted in the identification of 40 differentially expressed proteins, including four cytoskeletal proteins, ten chaperone and folding proteins, seven metabolic enzymes and seven other proteins of varied functions. The induction of seven ER chaperones and foldases is a solid indication of the onset of the unfolded protein response, which is triggered by cellular and ER stresses, many of which occur during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes that a change in the energy metabolism likely occurred as culture conditions degraded and apoptosis advanced. By identifying the intracellular changes during cultivation, this study provides a foundation for optimizing cell line-specific cultivation processes, prolonging longevity and maximizing protein production.     

Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice

We examined the effects of lactate on the enzymatic activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) in various mouse tissues. Our results showed that lactate inhibited PFK activity in all the analyzed tissues. This inhibitory effect was observed in skeletal muscle even in the presence of insulin. Lactate directly inhibited the phosphorylation of PFK tyrosine residues in skeletal muscle, an important mechanism of the enzyme activation. Moreover, lactate indirectly inhibited HK activity, which resulted from its cellular redistribution, here attributed to alterations of HK structure. PK activity was not affected by lactate. The activity of HK and PFK is directly related to glucose metabolism. Thus, it is conceivable that lactate exposure can induce inhibition of glucose consumption in tissues.     

Ezrin and HNRNP expression correlate with increased virus release rate and early onset of virus‐induced apoptosis of MDCK suspension cells

With the aim to adapt high‐yield adherent cell lines to suspension growth, Madin Darby canine kidney (MDCK) suspension cells were developed recently that achieved comparable influenza virus yields despite an early induction of apoptosis compared to the parental adherent cell line. For both cell lines, a comprehensive study under comparable infection conditions is performed comprising information on: time course of viral infection, antiviral state of cells, virus‐induced apoptosis, and virus‐induced cellular protein expression for early and late infection with influenza A/PuertoRico/8/34 H1N1. The proteomic analysis is performed with 2D differential gel electrophoreses followed by mass spectrometry. Based on flow cytometric data and on the differential expression of various stress and apoptosis‐related proteins, the earlier onset of virus‐induced apoptosis is confirmed for suspension cells. Surprisingly, the data indicated an increased virus release rate for suspension cells. These observations correlate with an increased expression of the apical marker protein ezrin, known to play a role in influenza‐induced cytoskeletal rearrangement, and the differential expression of heterogeneous nuclear ribonucleoproteins, known to bind actively influenza viral proteins and play a central role in regulating gene expression. Based on these findings, additional studies towards the design of MDCK suspension cells with further increase in influenza virus yields will be performed.     

Heterogeneity of glycolytic oscillatory behaviour in individual yeast cells

There are many examples of oscillations in biological systems and one of the most investigated is glycolytic oscillations in yeast. These oscillations have been studied since the 1950s in dense, synchronized populations and in cell-free extracts, but it has for long been unknown whether a high cell density is a requirement for oscillations to be induced, or if individual cells can oscillate also in isolation without synchronization. Here we present an experimental method and a detailed kinetic model for studying glycolytic oscillations in individual, isolated yeast cells and compare them to previously reported studies of single-cell oscillations. The importance of single-cell studies of this phenomenon and relevant future research questions are also discussed.     

Developmental changes of the glycolytic enzymes in the human fetal heart

The ontogeny of hexokinase, phosphofructokinase, phosphoglucoisornerase, aldolase, pyruvate kinase and lactate dehydrogenase activities which are associated with glycolysis, an important energy yielding process, has been studied in human fetal heart for periods ranging from 13 weeks to above 33 weeks of gestation. Hexokinase, phosphoglucoisomerase and pyruvate kinase activities show similar developmental profiles exhibiting maximum activity at 25–28 weeks ofgestation. Phosphofructokinase activity, on the other hand, shows a minimum at this period and exhibits a peak value at early stages (13–16 weeks of gestation). Though considerable activity for aldolase is observed at an early period, it declines thereafter, but again increases in the later period. The probable role and correlations of these glycolytic enzymes with energy demand and general functional development in human fetal heart in ontogeny are evaluated.     

Candidate autoantigens identified by mass spectrometry in early rheumatoid arthritis are chaperones and citrullinated glycolytic enzymes

Introduction  

The aim of our study was to identify new early rheumatoid arthritis (RA) autoantibodies.     

Purification and identification of activating enzymes of CS-0777, a selective sphingosine 1-phosphate receptor 1 modulator, in erythrocytes

CS-0777 is a selective sphingosine 1-phosphate (S1P) receptor 1 modulator with potential benefits in the treatment of autoimmune diseases, including multiple sclerosis. CS-0777 is a prodrug that requires phosphorylation to an active S1P analog, similar to the first-in-class S1P receptor modulator FTY720 (fingolimod). We sought to identify the kinase(s) involved in phosphorylation of CS-0777, anticipating sphingosine kinase (SPHK) 1 or 2 as likely candidates. Unlike kinase activity for FTY720, which is found predominantly in platelets, CS-0777 kinase activity was found mainly in red blood cells (RBCs). N,N-Dimethylsphingosine, an inhibitor of SPHK1 and -2, did not inhibit CS-0777 kinase activity. We purified CS-0777 kinase activity from human RBCs by more than 10,000-fold using ammonium sulfate precipitation and successive chromatography steps, and we identified fructosamine 3-kinase (FN3K) and fructosamine 3-kinase-related protein (FN3K-RP) by mass spectrometry. Incubation of human RBC lysates with 1-deoxy-1-morpholinofructose, a competitive inhibitor of FN3K, inhibited ～10% of the kinase activity, suggesting FN3K-RP is the principal kinase responsible for activation of CS-0777 in blood. Lysates from HEK293 cells overexpressing FN3K or FN3K-RP resulted in phosphorylation of CS-0777 and structurally related molecules but showed little kinase activity for FTY720 and no kinase activity for sphingosine. Substrate preference was highly correlated among FN3K, FN3K-RP, and rat RBC lysates. FN3K and FN3K-RP are known to phosphorylate sugar moieties on glycosylated proteins, but this is the first report that these enzymes can phosphorylate hydrophobic xenobiotics. Identification of the kinases responsible for CS-0777 activation will permit a better understanding of the pharmacokinetics and pharmacodynamics of this promising new drug.     

FHL2‐driven molecular network mediated Septin2 knockdown inducing apoptosis in mesangial cell

The apoptosis of mesangial cells (MCs) plays a critical role in the pathological progress of MesPGN. Septin2, a filamentous GTPase, is implicated in the apoptotic progress of MCs in the rat MesPGN model. However, the molecular mechanism of SEPT2 in MCs apoptosis is not clear. Here, we present the FHL2‐driven molecular network as the main mechanism of SEPT2‐mediated rat primary MCs apoptosis. First, we proved that the expression of FHL2 and Septin2 were closely related with MCs apoptosis in anti‐Thy1 nephritis model. Then, it was found that FHL2 was a new interaction protein of Septin2 and Septin2 knockdown could induce MC apoptosis by FHL2‐mediatied signal pathways including p‐ERK1 and p‐AKT. We applied label‐Free quantitative proteomics to identify the mechanism of Septin2/FHL2‐regulated apoptosis. Bioinformatics analysis revealed that FHL2‐driven molecular network composed of biological functions including glycolysis, oxidative stress, ribonucleotide metabolism, actin cytoskeleton regulation, and signaling pathway, was the main mechanism of SETP2‐mediated apoptosis. Furthermore, we showed that the effect of Septin2 knockdown on MC apoptosis could be alleviated by the overexpression of FHL2. Overall, this study illustrated the FHL2‐driven molecular network controlling SEPT2‐mediated apoptosis in MCs and their potential roles in mesangial proliferative nephritis.     

饲料碳水化合物水平对南方鲇幼鱼餐后糖酵解酶活性及血糖浓度的影响

配制含0%、15%和30%糊化玉米淀粉的等能饲料分别作为对照、中水平和高水平碳水化合物(CHO)饲料,以体重为50.5±0.6g的南方鲇幼鱼为实验对象,在水温27.5±0.5℃的条件下以对照饲料驯化15d后,分别测定了以三种饲料投喂的南方鲇在餐后3、61、2、24和48h的己糖激酶(HK)、葡萄糖激酶(GK)和磷酸果糖激酶(PFK-1)的活性和血糖水平。结果发现,中、高水平CHO组HK活性在餐后24h时显著高于对照组(P<0.05),其余时间点各组间无显著差异;GK活性随CHO水平增加而增加,但其活性的绝对值远低于HK;PFK-1活性在各组间及同组内各时间点均无显著差异。通过分析认为,南方鲇体内葡萄糖磷酸化主要由HK催化,但其活性受饲料CHO水平的影响不明显;GK活性受饲料CHO的诱导而增高,使葡萄糖磷酸化加速,但最大增幅不超过30%;此外,催化果糖-6-磷酸进一步转化为果糖-1,6-二磷酸的PFK-1活性不受饲料CHO水平的影响,因此糖酵解过程的这两个代谢环节均不能在鱼体吸收高糖营养后有效地加速,这应当是该肉食性鱼类在高水平CHO营养条件下产生高血糖积累的重要原因。实验结果表明,血糖变化呈现先升高后降低的趋势,中水平和高水平CHO组的血糖峰值均出现在餐后12h,这作为在该类研究中设定血液取样时间的实验依据。     

Human mature erythroblasts are resistant to apoptosis

Apoptosis plays an important role in red blood cell development, notably by regulating the fate of early erythroid progenitors. We show here that, by contrast, mature erythroblasts are resistant to apoptosis. Treatment of these cells with several apoptosis-inducing agents failed to trigger caspase activation and oligonucleosomal DNA fragmentation. Interestingly, we find that cytochrome c levels are dramatically reduced even though the cells contain mitochondria. Supplementation of cytosolic extracts from mature erythroblasts with cytochrome c, however, did not rescue caspase activation. This was not due to the presence of inhibitors of apoptosis, as these proteins were also missing in these cells. We also show that cytochrome c depletion is a normal event during erythroblast differentiation, which follows transient, developmentally induced caspase activation and correlates with the loss of response to cytokine withdrawal or drug-induced apoptosis. Our data therefore suggest that erythroblasts acquire resistance to apoptosis during maturation through the developmentally induced depletion of cytochrome c and other crucial regulators of the apoptotic machinery.     

Benzobijuglone, a novel cytotoxic compound from Juglans mandshurica, induced apoptosis in HeLa cervical cancer cells

A new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ), isolated from Juglans mandshurica by bioassay-guided fractionation, showed cytotoxic activity against HeLa cell line. Its chemical structure was determined by NMR and HREIMS spectra. In this paper, its ability to induce apoptosis in HeLa cells was studied for the first time. After treated with HMBBJ, the growth of HeLa cells was inhibited and cells displayed typical morphological apoptotic characteristics. Data from flow cytometry analysis showed that the HeLa cell cycle was arrested in the G2/M phase by HMBBJ, and the apoptotic rate of HeLa cells increased in a dose-dependent manner. Meanwhile, HMBBJ increased the expression of caspase-8, -3 and Bax, decreased the expression of Bcl-2, and lowered the ΔΨ_m. These findings reveal that HMBBJ could efficiently induce HeLa cells apoptosis through mitochondria dependent pathway and activation of the caspase cascade, and it may be a potential chemotherapeutic candidate for the treatment of cancer.     

Monitoring CHO cell cultures: Cell stress and early apoptosis assessment by mass spectrometry

Mammalian cells, especially CHO (Chinese Hamster Ovary), are an important host for the production of biopharmaceuticals. Early detection of cellular stress and the onset of apoptosis, ultimately leading to a reduced viability of the culture, are important with respect to process development and monitoring.     

Fibroblasts from the muscles of Duchenne muscular dystrophy patients are resistant to cell detachment apoptosis

Extracellular matrix (ECM) proteins, including collagen and growth factors, are greatly increased in tissue fibrosis and mainly secreted by fibroblasts. We previously demonstrated that muscle-derived fibroblasts from Duchenne muscular dystrophy (DMD) patients have a profibrotic phenotype, that includes significantly reduced expression of tissue inhibitor of metalloprotease 3 (TIMP-3) compared to control. Since TIMP-3 induces apoptosis in various cell types, we hypothesized increased resistance of DMD fibroblasts to apoptosis. To address this, we evaluated apoptotic nuclei, caspase 3, caspase 3 substrate expression, and migration and adhesion properties of muscle-derived fibroblasts, after applying different apoptosis-inducing treatments. We found that DMD fibroblasts were less susceptible to cell death, more adhesive, and had greater tendency to migrate than control fibroblasts — findings further supported by alterations in FAK and ERK/MAPK expression. Resistance to apoptosis and greater adhesion are likely to contribute to muscle fibrosis so a pharmacological treatment that targets dysregulated pathways involved in cell detachment apoptosis (anoikis) may limit the progressive fibrotic remodeling characteristic of DMD.     

Antizyme1基因转染对K562细胞增殖与凋亡的影响

研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷（ As2O3）诱导凋亡时的影响。方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体。脂质体法转染K562细胞,通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系。采用不同浓度的As2O3处理细胞,通过MTT法检测细胞增殖,流式细胞术分析细胞周期及凋亡变化。并通过RT-PCR方法检测antiyme1转染对cyclin D1和survivin基因表达的影响。结果：获得稳定表达antizyme1的K562-AZ1m细胞株后,其增殖能力明显减慢。CyclinD1基因表达降低,细胞主要停滞于G0/G1期。在 As2O3的诱导作用下,细胞凋亡增多,survivin基因表达降低。结论：AZ1基因能够抑制K562细胞增殖,通过对cyclinD1的负调控使细胞周期停滞于G0/G1期。并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用     

FAM129B/MINERVA, a novel adherens junction-associated protein, suppresses apoptosis in HeLa cells

A recent proteomics study identified FAM129B or MINERVA as a target of the MAP kinase (Erk1/2) signaling cascade in human melanoma cells. Phosphorylation of the protein was found to promote cell invasion and the dissociation of the protein from the cell-cell junctions. Suppression of apoptosis during metastasis is a prerequisite for the survival and spread of cancer cells. During apoptosis, the adherens junctions are disassembled as the dying cell retracts, and new contacts are formed between normal neighboring cells. In this study, we show that FAM129B was cytosolic in exponentially growing HeLa cells but was translocated to the adherens junctions where it colocalized with β-catenin whenever contact between two or more cells was established. Silencing the FAM129B gene expression by specific siRNAs did not induce apoptosis or inhibit the growth of HeLa cells. However, when apoptosis was induced by exposure to TNFα/cycloheximide or other apoptotic signaling molecules, the onset of apoptosis was accelerated 3-4-fold when FAM129B was depleted. Annexin V binding, the inactivation of the DNA repair enzyme, poly(ADP-ribose) polymerase, and the activation of the caspases occurred more rapidly in the cells lacking FAM129B. The rapid induction of apoptosis in FAM129B knockdown cells was reversed by co-transfection with recombinant FAM129B, indicating that its effect on apoptosis was specific. As apoptosis proceeded, FAM129B was degraded and disappeared from the plasma membrane. Thus, one crucial facet of the mechanism by which FAM129B promotes cancer cell invasion is likely to be the suppression of apoptosis.     

Design,synthesis and biological evaluation of pyrimidine derivatives as novel CDK2 inhibitors that induce apoptosis and cell cycle arrest in breast cancer cells

Cyclin-dependent kinase 2 (CDK2) plays a key role in eukaryotic cell cycle progression which could facilitate the transition from G1 to S phase. The dysregulation of CDK2 is closely related to many cancers. CDK2 is utilized as one of the most studied kinase targets in oncology. In this article, 24 benzamide derivatives were designed, synthesized and investigated for the inhibition activity against CDK2. Our results revealed that the compound 25 is a potent CDK2 inhibitor exhibiting a broad spectrum anti-proliferative activity against several human breast cancer cells. Additionally, compound 25 could block cell cycle at G0 or G1 and induce significant apoptosis in MDA-MB-468 cells. These findings highlight a rationale for further development of CDK2 inhibitors to treat human breast cancer.