eIF2α kinases control chalone production in Dictyostelium discoideum

Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high-molecular-weight complex that reversibly inhibits cell proliferation, but not growth, via cell surface receptors and a signaling pathway that includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2-type eukaryotic initiation factor 2 α subunit (eIF2α) kinases, proteins that phosphorylate the translational initiation factor eIF2α and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due to a general inhibition of translation. Instead, it is due to enhanced production of a secreted factor(s). Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that contain a nonphosphorylatable eIF2α. We conclude that production of the chalones CfaD and AprA is translationally regulated by eIF2α phosphorylation. Both proteins are upregulated at the culmination of development, and this enhanced production is lacking in a strain that possesses a nonphosphorylatable eIF2α.     

α-Mannosidase-2: A developmentally regulated enzyme in Dictyostelium discoideum

A previously undected isozyme of α-mannosidase was observed in several independent mutant strains of Dictyostelium discoideum selected for the absence of the major isozyme, α-mannosidase-1. The activity in the mutant strains, α-mannosidase-2, differs from the major isozyme with respect to pH optimum, substrate affinity, sensitivity to inhibition by l-cysteine, and is particulate bound. The enzyme can be solubilized by treatment of the extract with nonionic detergents. α-Mannosidase-2 begins to accumulate only after 12 hr of development and reaches a peak specific activity of about a tenth of that of α-mannosidase-1 during culmination. The increase in specific activity of α-mannosidase-2 is blocked by either cycloheximide or actinomycin D, drugs known to inhibit protein and RNA synthesis, respectively, and probably results from accumulation of de novo synthesized enzyme. α-Mannosidase-2, therefore, provides a convenient marker enzyme for biochemical differentiation during the pseudo-plasmodial stage.     

Mitochondrial tRNA 5′-Editing in Dictyostelium discoideum and Polysphondylium pallidum

Mitochondrial tRNA (mt-tRNA) 5′-editing was first described more than 20 years ago; however, the first candidates for 5′-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5′-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5′-editing in D. discoideum with 5′-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5′-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5′-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species.     

Dictyostelium discoideum—a model for many reasons

The social amoeba or cellular slime mould Dictyostelium discoideum is a “professional” phagocyte that has long been recognized for its value as a biomedical model organism, particularly in studying the actomyosin cytoskeleton and chemotactic motility in non-muscle cells. The complete genome sequence of D. discoideum is known, it is genetically tractable, readily grown clonally as a eukaryotic microorganism and is highly accessible for biochemical, cell biological and physiological studies. These are the properties it shares with other microbial model organisms. However, Dictyostelium combines these with a unique life style, with motile unicellular and multicellular stages, and multiple cell types that offer for study an unparalleled variety of phenotypes and associated signalling pathways. These advantages have led to its recent emergence as a valuable model organism for studying the molecular pathogenesis and treatment of human disease, including a variety of infectious diseases caused by bacterial and fungal pathogens. Perhaps surprisingly, this organism, without neurons or brain, has begun to yield novel insights into the cytopathology of mitochondrial diseases as well as other genetic and idiopathic disorders affecting the central nervous system. Dictyostelium has also contributed significantly to our understanding of NDP kinase, as it was the Dictyostelium enzyme whose structure was first determined and related to enzymatic activity. The phenotypic richness and tractability of Dictyostelium should provide a fertile arena for future exploration of NDPK’s cellular roles.     

Pacemakers in aggregation fields of Dictyostelium discoideum: does a single cell suffice?

In this paper we address the following question: can a single cell of the cellular slime mold Dictyostelium discoideum serve as a pacemaker for the aggregation phase? Whether or not this is possible is determined by the relative importance of cyclic AMP production due to self-stimulation as compared to diffusion of cyclic AMP away from the cell and extracellular degradation. We determine the conditions under which a single cell on an infinite place can emit periodic signals of cyclic AMP using a model developed previously for signal relay and adaptation in Dictyostelium. Elsewhere it has been shown that this model provides an accurate representation of the stimulus-response behavior of Dictyostelium for a variety of experimental conditions.     

Immunological relationship between β-N-acetylglucosaminidase and Proteinase I from Dictyostelium discoideum

Purified β-N-acetylglucosaminidase and purified Proteinase I, both isolated from the cellular slime mold, Dictyostelium discoideum, were shown to be immunologically cross-reactive with antiserum against the proteinase. A peptide was isolated from a papain digest of Proteinase I that completely inhibited the immunoprecipitation of β-N-acetylglucosaminidase, but only partially inhibited the immunoprecipitation of Proteinase I. This peptide inhibitor contained a high concentration of N-acetylglucosamine-1-phosphorylserine. It was proposed that these phosphoryl moieties might represent a common antigenic determinant in the two enzymes.     

The α-glucosidases of Dictyostelium discoideum: I. Identification and characterization

We have identified four isozymes of α-glucosidase in the cellular slime mold, Dictyostelium discoideum. The isozymes can be distinguished by their physical and enzymatic properties. α-Glucosidase-1, α-glucosidase-2, and α-gluocosidase-3 are all present in vegetative cells, while α-glucosidase-4 is present only after the cells have proceeded through aggregation. Three of the four enzymes, α-glucosidase-1, α-glucosidase-3, and α-glucosidase-4, have acidic pH optima of 3.5, 2.2, and 4.0, respectively. In contrast, α-glucosidase-2 has a neutral pH optimum, 7.25. α-Glucosidase-1, α-glucosidase-2, and α-glucosidase-3 are distinguishable by electrophoresis in native polyacrylamide gels. α-Glucosidase-4 comigrates with α-glucosidase-2 on native gels but they can be resolved by isoelectric focusing. The isozymes also differ with respect to affinity for the substrates p-nitrophenyl-α-d-glucoside and 4-methyl-umbelliferyl-α-d-glucopyranoside and the relative maximal rates of hydrolysis of these substrates. α-Glucosidases-1, -2, and -4 have apparent K_m's in the millimolar range while the apparent K_m of α-glucosidase-3 for p-nitrophenyl-α-d-glucoside is much higher. This may suggest that isozyme 3 is an endoglycosidase or may have greater affinity for other sugar substrates. α-Glucosidase-1 is the major isozyme in vegetative cells.     

盘基网柄菌(Dictyostelium discoideum)细胞的分化及其调控

本文综述了盘基网柄菌(Dictyostelium dis-coideum)发育过程中细胞类型的诱导和分化,细胞外cAMP及其四种位于细胞表面的受体及PKA(蛋白激酶A)、GSK-3(糖原合成酶激酶)和STATa等在网柄菌发育过程中的作用。     

ΔNp63α promotes cellular quiescence via induction and activation of Notch3

Genetic analysis of TP63 indicates that ΔNp63 isoforms are required for preservation of self-renewing capacity in the stem cell compartments of diverse epithelial structures; however, the underlying cellular and molecular mechanisms remain incompletely defined. Cellular quiescence is a common feature of adult stem cells that may account for their ability to retain long-term replicative capacity while simultaneously limiting cellular division. Similarly, quiescence within tumor stem cell populations may represent a mechanism by which these populations evade cytotoxic therapy and initiate tumor recurrence. Here, we present evidence that ΔNp63α, the predominant TP63 isoform in the regenerative compartment of diverse epithelial structuresm, promotes cellular quiescence via activation of Notch signaling. In HC11 cells, ectopic ΔNp63α mediates a proliferative arrest in the 2N state coincident with reduced RNA synthesis characteristic of cellular quiescence. Additionally, ΔNp63α and other quiescence-inducing stimuli enhanced expression of Notch3 in HC11s and breast cancer cell lines, and ectopic expression of the Notch3 intracellular domain (N3^ICD) was sufficient to cause accumulation in G₀/G₁ and increased expression of two genes associated with quiescence, Hes1 and Mxi1. Pharmacologic inhibition of Notch signaling or shRNA-mediated suppression of Notch3 were sufficient to bypass quiescence induced by ΔNp63α and other quiescence-inducing stimuli. These studies identify a novel mechanism by which ΔNp63α preserves long-term replicative capacity by promoting cellular quiescence and identify the Notch signaling pathway as a mediator of multiple quiescence-inducing stimuli, including ΔNp63α expression.Key words: p63, Notch, quiescence, stem cell     

ΔNp63α promotes cellular quiescence via induction and activation of Notch3

Genetic analysis of TP63 indicates that ΔNp63 isoforms are required for preservation of self-renewing capacity in the stem cell compartments of diverse epithelial structures; however, the underlying cellular and molecular mechanisms remain incompletely defined. Cellular quiescence is a common feature of adult stem cells that may account for their ability to retain long-term replicative capacity while simultaneously limiting cellular division. Similarly, quiescence within tumor stem cell populations may represent a mechanism by which these populations evade cytotoxic therapy and initiate tumor recurrence. Here, we present evidence that ΔNp63α, the predominant TP63 isoform in the regenerative compartment of diverse epithelial structuresm, promotes cellular quiescence via activation of Notch signaling. In HC11 cells, ectopic ΔNp63α mediates a proliferative arrest in the 2N state coincident with reduced RNA synthesis characteristic of cellular quiescence. Additionally, ΔNp63α and other quiescence-inducing stimuli enhanced expression of Notch3 in HC11s and breast cancer cell lines, and ectopic expression of the Notch3 intracellular domain (N3^ICD) was sufficient to cause accumulation in G₀/G₁ and increased expression of two genes associated with quiescence, Hes1 and Mxi1. Pharmacologic inhibition of Notch signaling or shRNA-mediated suppression of Notch3 were sufficient to bypass quiescence induced by ΔNp63α and other quiescence-inducing stimuli. These studies identify a novel mechanism by which ΔNp63α preserves long-term replicative capacity by promoting cellular quiescence and identify the Notch signaling pathway as a mediator of multiple quiescence-inducing stimuli, including ΔNp63α expression.     

Prostaglandin F2α promotes ovulation in prepubertal heifers

The objective was to determine the effects of exogenous prostaglandin F_2α (PGF), with or without progesterone treatment, on first ovulation in prepubertal heifers. We tested the hypothesis that PGF has a luteolysis-independent ovulatory effect in cattle. Crossbred Angus heifers (12 to 14 mo old, 250 kg body weight, and an average body condition score of 3 out of 5) were examined by transrectal ultrasonography on two occasions, 11 days apart. Heifers in which a CL was not detected at either examination were considered prepubertal. Heifers were assigned randomly to three experimental groups: (1) PG group (N = 14); heifers were treated with a PGF analog (500 μg cloprostenol im) 5 days after the emergence of a spontaneous (i.e., naturally occurring, noninduced) follicular wave; (2) PPG group (N = 12); heifers were given an intravaginal progesterone-releasing insert (CIDR; Pfizer Animal Health, Montreal, QC, Canada), and a follicular wave was induced with 50 mg of progesterone + 2 mg of estradiol benzoate im, and a PGF analog was given at the time of CIDR removal, on Day 5 of the follicular wave (on average, 8.6 ± 0.5 days after CIDR insertion); and (3) control group heifers were given no treatment (N = 14). Heifers were examined daily by transrectal ultrasonography from the start of the experiment to confirmation that ovulation had occurred, or to 5 days after PGF injection (PG and PPG groups) or until dominant follicles of the next follicular wave reached 8 mm (control group). The percentage of heifers that ovulated within 10 days after wave emergence was higher in PPG (10/12; 83.3%) and PG (11/14; 78.5%) groups than in control (1/14; 7.1%; P < 0.0001). Ovulations occurred 69.6 ± 6 h and 93.8 ± 5 h after PGF treatment in PPG and in PG groups, respectively, whereas only one heifer in the control group ovulated 96 h after Day 5 of follicular wave (P = 0.13). In summary, PGF treatment was associated with ovulation in prepubertal heifers whether or not exogenous progesterone was used as a pretreatment. The hypothesis that PGF will induce ovulation by a luteolysis-independent mechanism was supported.     

The induction of autophagy in isolated insect fat body by β-ecdysone

The fat body in Calpodes undergoes sequential organelle specific autophagy as a first step in the cell remodeling process necessary for metamorphosis to the pupa. This autophagy begins at about 36 hr before pupation and coincides with a critical period after which an isolated abdomen will pupate without further influence from the prothoracic glands. This suggested that autophagy might be induced by ecdysone. Fat body taken before the critical period and cultured in a medium containing β-ecdysone undergoes autophagy. Fat body from the same animal maintained in hormone-free medium retains the pre-critical period morphology with no autophagy. Autophagy is therefore directly induced by β-ecdysone. Fat body taken soon after the critical period continues with the autophagic sequence in hormone-free medium. Therefore the entire autophagic sequence is induced and does not require the continuing presence of hormone. Protein storage granule formation and cell dissociation, which occur in fat body at metamorphosis, are also induced by β-ecdysone.     

Autologous blood transfusion promotes autophagy and inhibits hepatocellular carcinoma progression through HIF-1α signalling pathway

To explore the molecular mechanism of autologous blood transfusion promoting autophagy of hepatocellular carcinoma (HCC) cells and inhibiting the HCC progression through HIF-1α signalling pathway. This is a research paper. Rat hepatocellular carcinoma model and HepG2 cell model were built. The rats with HCC were conducted a surgery, and their blood was collected for detection to detect the recurrence and metastasis of the rats. Western blot was used to analysed the expression of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein. The apoptosis rate of HepG2 cells was detected by flow cytometry, and autophagosomes were observed by transmission electron microscopy. HIF-1α expression was measured by immunofluorescence assay. The expressions of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein were highest in model + autoblood group compared with the model group. HIF-1α content of model group was higher, but content of TP53, MDM2, ATG5 and ATG14 in the model group is the second. The highest apoptosis rate was found in HepG2 + autoblood group. The number of autophagosomes in HepG2 + autoblood was obviously larger than that of HepG2 + autoblood + inhibitor. HIF-1α expression of immunofluorescence assay showed that high expression of HIF-1α was clearly observed in HepG2 and HepG2 + autoblood group from confocal observation. However, there was no HIF-1α protein expression in HepG2 + autoblood + inhibitor group. The migration rate in HepG2 group, HepG2 + autoblood group and HepG2 + autoblood + inhibitor group was 85.71 ± 7.38%, 14.36 ± 6.54% and 61.25 ± 5.39%, respectively. Autologous blood transfusion promotes autophagy of HCC cells through HIF-1α signalling pathway, which further inhibits HCC migration and erosion.     

Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum

In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms.     

盘基网柄菌(Dictyostelium discoideum)吞噬作用中肌动蛋白多聚化及其调节

肌动蛋白是盘基网柄菌(Dictyostelium discoideum)细胞吞噬过程中的关键组分,通过其细胞内的定位和多聚化形式在确定的时间和地点连接特定的分子,使吞噬过程得以完成。profilin是肌动蛋白多聚化的重要调节分子,在磷脂酰肌醇信号转导与细胞骨架相交处起关键作用。许多小分子G蛋白参与细胞骨架调节,CAP蛋白是两者间重要连接分子。所以,吞噬作用是细胞内诸分子协同作用的结果。     

PKA在盘基网柄菌(Dictyostelium discoideum)多细胞发育中的作用

在盘基网柄菌(Dictyosteliumdiscoideum)多细胞发育中,蛋白激酶A(proteinkinaseA,PKA)发挥多重作用.细胞聚集阶段,PKA调节腺苷酰环化酶的活性,中转cAMP,诱导dut、pdi等一些发育早期的基因表达;参与启动聚集后的细胞分化和形态构成,增强GBF活性,激活前孢子细胞特有基因的表达;它还精密调控前柄细胞特有基因ecmB的表达,准确启动拔顶发育,诱导孢柄和孢子的成熟.子实体形成后,PKA又是维持孢子休眠和保证孢子有效萌发的必需因子.在PKA调控下,盘基网柄菌有条不紊地完成整个发育过程.     

盘基网柄菌(Dictyostelium discoideum)突变型细胞allC细胞周期异常的研究

细胞计数和细胞倍增时间计算的结果表明allC细胞的倍增时间为2.36h,仅为KAx-3细胞倍增时间的1/3。为了探究allC细胞倍增时间大幅度缩短、细胞周期异常的原因我们采用流式细胞术测定两种细胞的细胞周期,并结合实时荧光定量PCR技术测定cycB1和cdk1基因的相对表达量的比值。结果表明,16h突变型allC细胞处于G2期的数目(1.51%)显著少于KAx-3细胞(16.61%)。allC细胞和KAx-3细胞的细胞周期素B1(cyclinB1)cycB1基因相对表达量分别是2.5和0.25,两者相差10倍。这些数据表明,两种类型细胞中G2期的差异十分明显,cyclinB1的相对表达量也存在显著差异。提示cyclinB1的过表达可能在一定程度上影响allC细胞的细胞周期正常的调控机制,与突变细胞的G2期异常有一定关系。     

MiR-141–3p promotes hypoxia-induced autophagy in human placental trophoblast cells

Preeclampsia (PE) is a pregnancy-specific disorder and a significant contributor to maternal, fetal and neonatal morbidity and mortality worldwide. Its pathogenesis is generally accepted as insufficient trophoblast invasion of the maternal endometrium and inadequate remodeling of the maternal spiral arteries. These impairments lead to elevated levels of hypoxia and oxidative stress. Autophagy has become a highly researched field in obstetrics, and this process may be essential for preimplantation development beyond the four- and eight-cell stages, and for blastocyst survival, extra-villous trophoblast functions, invasion and vascular remodeling. Several studies have shown that autophagy activation, shown by an increase in autophagy vacuoles or microtubule-associated protein 1 A/1B-light chain 3 (LC3) dots, was more common in PE than in normal pregnancy. Thus, changes in autophagic status are seen in preeclamptic placentas. MicroRNA-141–3p (miR-141–3p), a multifunctional miRNA, is involved in a variety of physiological and pathological processes, including PE and autophagy. However, the influence of miR-141–3p on autophagy regulation in trophoblast cells has yet to be described. Therefore, the objective of our study was to investigate the role of miR-141–3p in autophagy induced by hypoxia in human placental trophoblast cells. Our results found that hypoxia induced autophagy in trophoblast cells and dramatically elevated the expression of miR-141–3p. Overexpression of miR-141–3p improved autophagic activity, whereas low expression of miR-141–3p inhibited autophagic activity. Therefore, our data demonstrated that miR-141–3p promoted hypoxia-induced autophagy in placental trophoblast cells, which may be related to the development of preeclampsia.