Differing mechanisms of cAMP- versus seawater-induced oocyte maturation in marine nemertean worms I. The roles of serine/threonine kinases and phosphatases

Unlike in most animals, oocytes of marine nemertean worms initiate maturation (=germinal vesicle breakdown, GVBD) following an increase, rather than a decrease, in intraoocytic cAMP. To analyze how serine/threonine (Ser/Thr) kinase cascades involving mitogen-activated protein kinase (MAPK), maturation-promoting factor (MPF), cAMP-dependent protein kinase (PKA), and phosphatidylinositol 3-kinase (PI3K) regulate nemertean GVBD, oocytes of Cerebratulus sp. were treated with pharmacological modulators and stimulated with cAMP-elevating drugs or seawater (SW) alone. Both cAMP elevators and SW triggered GVBD while activating MAPK, its target p90Rsk, and MPF. Similarly, neither cAMP- nor SW-induced GVBD was affected by several Ser/Thr phosphatase inhibitors, and both stimuli apparently accelerated GVBD via a MAPK-independent, PI3K-dependent mechanism. However, inhibitors of Raf-1, a kinase that activates MAPK kinase, blocked GVBD and MAPK activation during SW-, but not cAMP-induced maturation. In addition, MPF blockers more effectively reduced GVBD and MAPK activity in SW versus in cAMP-elevating treatments. Moreover, the two maturation-inducing stimuli yielded disparate patterns of PKA-related MAPK activations and phosphorylations of putative PKA substrates. Collectively, such findings suggest that in maturing oocytes of Cerebratulus sp., Ser/Thr kinase cascades differ during cAMP- versus SW-induced GVBD in several ways, including MAPK activation modes, MPF-feedback loops, and PKA-related signaling pathways. Additional differences in cAMP- versus SW-induced oocyte maturation are also described in the accompanying study that deals with the roles of tyrosine kinase signaling during GVBD.     

Germinal vesicle breakdown is not fully dependent on MAPK activation in maturing oocytes of marine nemertean worms

Previously, it has been shown that oocytes of marine nemertean worms resume meiosis and undergo germinal vesicle breakdown (GVBD) following treatment with either natural seawater (NSW), or the neurohormone serotonin (5-hydroxytryptamine or 5-HT). In this investigation of the nemerteans Cerebratulus lacteus and Cerebratulus sp., immunoblots and kinase assays were used to compare the roles of two regulatory kinases: mitogen-activated protein kinase (MAPK) and Cdc2/cyclin B (referred to as maturation promoting factor or MPF). Based on such analyses, an ERK (extracellular signal regulated kinase) type of MAPK was found to be activated concurrently with Cdc2/cyclin B during NSW- and 5-HT-induced maturation. MAPK activation occurred prior to GVBD and seemed to be controlled primarily by phosphorylation rather than de novo protein synthesis. Inhibition of MAPK signaling by U0126 was capable of delaying but not permanently blocking Cdc2/cyclin B activation and GVBD in 5-HT treated oocytes and subsets of NSW-treated oocytes. Collectively such data indicated that GVBD is not fully dependent on MAPK activation, since Cdc2/cyclin B can apparently be activated by MAPK-independent mechanism(s) in maturing nemertean oocytes.     

The reverse, but coordinated, roles of Tor2 (TORC1) and Tor1 (TORC2) kinases for growth, cell cycle and separase-mediated mitosis in Schizosaccharomyces pombe

Target of rapamycin complexes (TORCs), which are vital for nutrient utilization, contain a catalytic subunit with the phosphatidyl inositol kinase-related kinase (PIKK) motif. TORC1 is required for cell growth, while the functions of TORC2 are less well understood. We show here that the fission yeast Schizosaccharomyces pombe TORC2 has a cell cycle role through determining the proper timing of Cdc2 Tyr15 dephosphorylation and the cell size under limited glucose, whereas TORC1 restrains mitosis and opposes securin-separase, which are essential for chromosome segregation. These results were obtained using the previously isolated TORC1 mutant tor2-L2048S in the phosphatidyl inositol kinase (PIK) domain and a new TORC2 mutant tor1-L2045D, which harbours a mutation in the same site. While mutated TORC1 and TORC2 displayed diminished kinase activity and FKBP12/Fkh1-dependent rapamycin sensitivity, their phenotypes were nearly opposite in mitosis. Premature mitosis and the G2-M delay occurred in TORC1 and TORC2 mutants, respectively. Surprisingly, separase/cut1-securin/cut2 mutants were rescued by TORC1/tor2-L2048S mutation or rapamycin addition or even Fkh1 deletion, whereas these mutants showed synthetic defect with TORC2/tor1-L2045D. TORC1 and TORC2 coordinate growth, mitosis and cell size control, such as Wee1 and Cdc25 do for the entry into mitosis.     

MAP kinases p38 and JNK are activated by the steroid hormone 1alpha,25(OH)2-vitamin D3 in the C2C12 muscle cell line

In chick skeletal muscle cell primary cultures, we previously demonstrated that 1alpha,25(OH)2-vitamin D3 [1alpha,25(OH)2D3], the hormonally active form of vitamin D, increases the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2, their subsequent translocation to the nucleus and involvement in DNA synthesis stimulation. In this study, we show that other members of the MAP kinase superfamily are also activated by the hormone. Using the muscle cell line C2C12 we found that 1alpha,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK. The immediately upstream mitogen-activated protein kinase kinases 3/6 (MKK3/MKK6) were also phosphorylated by the hormone suggesting their participation in p38 activation. 1Alpha,25(OH)2D3 was able to dephosphorylate/activate the ubiquitous cytosolic tyrosine kinase c-Src in C2C12 cells and studies with specific inhibitors imply that Src participates in hormone induced-p38 activation. Of relevance, 1alpha,25(OH)2D3 induced in the C2C12 line the stimulation of mitogen-activated protein kinase activating protein kinase 2 (MAPKAP-kinase 2) and subsequent phosphorylation of heat shock protein 27 (HSP27) in a p38 kinase activation-dependent manner. Treatment with the p38 inhibitor, SB203580, blocked p38 phosphorylation caused by the hormone and inhibited the phosphorylation of its downstrean substrates. 1Alpha,25(OH)2D3 also promotes the phosphorylation of c-jun N-terminal protein kinases (JNK 1/2), the response is fast (0.5-1 min) and maximal phosphorylation of the enzyme is observed at physiological doses of 1alpha,25(OH)2D3 (1 nM). The relative contribution of ERK-1/2, p38, and JNK-1/2 and their interrelationships in hormonal regulation of muscle cell proliferation and differentiation remain to be established.     

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.     

Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs

Full-length cDNAs encoding three forms of vitellogenin (Vg) were obtained from a liver cDNA library of estrogen-treated red seabream, Pagrus major. Two of the three Vg sequences had high homology with type-A and -B Vgs (VgA and VgB) of other teleosts. The third red seabream Vg was classified as a type-C or phosvitinless (Pvl) Vg due to its lack of a phosvitin (Pv) domain. Two Vg preparations (610 and 340 kDa) from blood serum of estradiol-treated fish were biochemically characterized. Analyses of precursor-product relationships by examination of N-terminal amino acid sequences verified cleavage of the 610 kDa Vg into a 540 kDa lipovitellin (Lv) and a 32 kDa beta'-component. Each of these yolk preparations comprising both VgA- and VgB-derived polypeptides. The 340 kDa Vg, which was immunologically verified to be a PvlVg, was accumulated by vitellogenic oocytes with no alterations to its native molecular mass. During oocyte maturation, the VgA- and VgB-derived yolk proteins were differentially processed, presumably to generate a pool of free amino acids for oocyte hydration or for allocation of specific types of nutrients, amino acids, and proteins, to the developing embryo. Conversely, the 340 kDa Vg-derived yolk protein is unlikely to contribute to oocyte hydration or diffusible nutrients since the molecule underwent only minor proteolytic nicking during oogenesis. The present study elucidates for the first time specific functions of three different forms of Vg and their product yolk proteins in a higher taxonomic group of marine teleosts that spawn pelagic eggs.     

Effect of protease inhibitors on protein degradation in rat hepatoma cells II. Effects on ornithine decarboxylase and tyrosine aminotransferase

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).     

Structural motifs in the maturation process of peptide hormones. The somatostatin precursor. I. A CD conformational study.

Synthetic peptides reproducing both the native domain around the dibasic cleavage site of prosomatostatin, and mutated sequences there of, previously assayed in site-directed mutagenesis experiments, have been studied by CD in different solvent systems, such as water, TFE/H2O, MeCN/H2O and aqueous SDS, in order to ascertain the ability of each solvent to stabilize secondary structural motifs. A combination of deconvolution methods and empirical calculations, that allow subtraction of the contributions due to unordered structures from the spectra, suggests that mainly two distinct families of ordered conformers containing alpha-helix and/or structurally different beta-turns are present in solution, the relative stability of the different conformers depending on the nature of the solvent. The presence of beta-turns is in line with a previous NMR study in DMSO and DMSO/H2O. Comparison of the CD spectra in aqueous SDS of peptides undergoing processing with a sequence not processed in vivo shows that only the latter possesses a stable and detectable alpha-helix population. This observation suggests that the structuration involving beta-turns but no alpha-helix, which was observed by CD both in SDS and organic solvent/H2O mixtures at high water contents, might be of biological significance. The similarity of this structuration to molecular models obtained from NMR data in DMSO and DMSO/H2O is discussed.     

The roles of NADPH oxidase and phospholipases A2 in oxidative and inflammatory responses in neurodegenerative diseases

Reactive oxygen species (ROS) are produced in mammalian cells through enzymic and non-enzymic mechanisms. Although some ROS production pathways are needed for specific physiological functions, excessive production is detrimental and is regarded as the basis of numerous neurodegenerative diseases. Among enzymes producing superoxide anions, NADPH oxidase is widespread in mammalian cells and is an important source of ROS in mediating physiological and pathological processes in the cardiovascular and the CNS. ROS production is linked to the alteration of intracellular calcium homeostasis, activation of Ca(2+)-dependent enzymes, alteration of cytoskeletal proteins, and degradation of membrane glycerophospholipids. There is evolving evidence that ROS produced by NADPH oxidase regulate neuronal functions and degrade membrane phospholipids through activation of phospholipases A(2) (PLA(2)). This review is intended to cover recent studies describing ROS generation from NADPH oxidase in the CNS and its downstream activation of PLA(2), namely, the group IV cytosolic cPLA(2) and the group II secretory sPLA(2). A major focus is to elaborate the dual role of NADPH oxidase and PLA(2) in mediating the oxidative and inflammatory responses in neurodegenerative diseases, including cerebral ischemia and Alzheimer's disease. Elucidation of the signaling pathways linking NADPH oxidase with the multiple forms of PLA(2) will be important in understanding the oxidative and degradative mechanisms that underline neuronal damage and glial activation and will facilitate development of therapeutic intervention for prevention and treatment of these and other neurodegenerative diseases.     

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti‐phosphotyrosine and anti‐phosphoserine antibodies following 2‐DE in conjunction with double channel laser‐induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans‐membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.     

The effects and mechanisms of SLC34A2 in tumorigenesis and progression of human non-small cell lung cancer

Background

SLC34A2 with highest expressions in lung, small intestine and kidney encoded a type 2b sodium-dependent phosphate transporter (NaPi-IIb). In lung, SLC34A2 only expressed in the apical membrane of type II alveolar epithelium cells (ATII cells) and played a pivotal role during the fetal lung development and embryonic development. ATII cells acting as multifunctional stem cells might transform into NSCLC after undergoing exogenous or endogenous factors. Increasing evidences showed that the genes performing critical roles during embryogenesis were also expressed during the development of cancer. In addition, recent research found the expression of SLC34A2 had a significant difference between the surgical samples of NSCLC and normal tissues, and SLC34A2 was down-regulated in lung adenocarcinoma cell line A549 and up-regulation expression of SLC34A2 could significantly inhibit cell viability and invasion of A549 in vitro. These results suggested SLC34A2 might play an important role in the development of NSCLC. However, the role of SLC34A2 in tumorigenesis and progression of NSCLC remains unknown.

Results

Our study found that SLC34A2 was also significantly down-regulated in 14/15 of examined NSCLC tissues. Moreover, we found that expressions of SLC34A2 were reduced in six NSCLC cell lines for the first time. Our result also revealed a dramatic inhibitory effects of SLC34A2 on cell growth, migration and invasion of several NSCLC cell lines. SLC34A2 also strongly inhibited tumor growth and metastasis ability in A549 subcutaneous tumor model and lung metastasis model, respectively. Further studies found that the suppressive effects of SLC34A2 on tumorigenesis and progression might be associated with the down-regulation of related protein in PI3K/Akt and Ras/Raf/MEK signal pathway.

Conclusions

For the first time, our data indicated that SLC34A2 could exert significantly suppressive effects on tumorigenesis and progression of NSCLC. SLC34A2 might provide new insights for further understanding the early pathogenesis of human NSCLC.     

Proliferative and morphological effects of endothelins in Schwann cells: roles of p38 mitogen-activated protein kinase and Ca(2+)-independent phospholipase A2.

The regulation of Schwann cell (SC) proliferation and morphology is critical to nerve homeostasis. We have previously reported that endothelins (ETs) regulate the activity of different effectors in SC including adenylyl cyclase, phospholipases C and A2 and mitogen-activated protein kinases (MAPKs). These effects imply a possible participation of ETs in the regulation of SC phenotype. We have now investigated the effects of endothelins on the proliferation and morphology of SC, and compared them with the responses to platelet-derived growth factor (PDGF), a known mitogen in these cells. Both endothelin-1 (ET-1) and PDGF increased the incorporation of [3H]thymidine and the proportion of SC in S and G2/M, with a concomitant decrease in the G0/G1 stage cells. Treatment with ET-1 produced rapid changes in the morphology of the SC, characterized by the appearance of cell spreading with shorter processes. The response to ET-1 was considered to represent a proliferative phenotype, in contrast to the effects of forskolin, which decreased [3H]thymidine incorporation in immortalized SC (iSC) and lead to a differentiated morphology with longer extensions. While both ET-1 and PDGF displayed a proliferative effect on SC, treatment with PDGF did not affect the morphology of these cells to a significant extent. A role for p38 MAPK and Ca(2+)-independent phospholipase A2 in the changes in morphology and proliferation of iSC driven by ET-1 was suggested by the effects of selective inhibitors of these pathways [SB202190 and HELSS, respectively]. The unique pattern of signaling pathways recruited by ET-1 and its combined effects on regulation of phenotype and proliferation of SC suggest an important role for this peptide during nerve degeneration/regeneration.     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. II. The platelet-derived growth factor A-chain contains a sequence that specifically binds heparin

Summary Synthetic oligopeptides were used to study the specificity of the interaction between heparin and platelet-derived growth factor (PDGF) in competition experiments. DNA synthesis in PDGF-dependent human arterial smooth muscle cell (hASMC) cultures was used as a biological tracer of PDGF activity. Oligo-108-124 (corresponding to amino acid residues 108-124 of the long PDGF A-chain isoform) had no effect on DNA synthesis in itself but competed at 10⁻¹⁰ M concentration effectively with PDGF for binding to heparin and released the block on thymidine incorporation induced by heparin. Poly-lysine-serine (lysine:serine ratio 3:1) was also effective but at a considerably higher concentration (10⁻⁶ M). Poly-arginine-serine did not compete with PDGF for heparin as deduced from the cell assay. This suggested that among basic amino acids, lysine was more important than arginine for heparin binding. Deletion of lysine residues 115 and 116 in Oligo-108-124 abolished its effect on the interaction between PDGF and heparin in the cell assay. Likewise, Oligo-69-84 (corresponding to the PDGF A-chain residues 69–84), with three lysine residues interrupted by a proline, was ineffective. In Oligo-108-124, the lysine residues are interrupted by an arginine. Our results suggested that the binding between PDGF and heparin is specific and that the amino acid sequence [-Lys¹¹⁵-Lys¹¹⁶-Arg¹¹⁷-Lys¹¹⁸-Arg¹¹⁹-] is of major importance. They do not however, exclude other domains of the PDGF A or B chains as additional binding sites for heparin nor do they exclude the possibility that heparin and the PDGF receptor share a common binding site.     

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. II. Interplay of local backbone conformational dynamics and long‐range hydrophobic interactions in hairpin formation

Two peptides, corresponding to the turn region of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus, consisting of residues 51–56 [IG(51–56)] and 50–57 [IG(50–57)], respectively, were studied by circular dichroism and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the β‐turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to those observed in the native protein. The formation of a turn is observed for both peptides in a broad range of temperatures (T = 283–323 K), which confirms the conclusion drawn from our previous studies of longer sequences from the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G (16, 14, and 12 residues), that the DDATKT sequence forms a nucleation site for formation of the β‐hairpin structure of peptides corresponding to the C‐terminal part of all the B domains of the immunoglobulin binding protein G. We also show and discuss the role of long‐range hydrophobic interactions as well as local conformational properties of polypeptide chains in the mechanism of formation of the β‐hairpin structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.