scholarly journals Ser149 Is Another Potential PKA Phosphorylation Target of Cdc25B in G2/M Transition of Fertilized Mouse Eggs

2011 ◽  
Vol 286 (12) ◽  
pp. 10356-10366 ◽  
Author(s):  
Jianying Xiao ◽  
Chao Liu ◽  
Junjie Hou ◽  
Cheng Cui ◽  
Didi Wu ◽  
...  
Keyword(s):  
Mouse Embryos ◽  
Negative Regulator ◽  
Phosphorylation Sites ◽  
Control Groups ◽  
Significant Difference ◽  
M Phase ◽  
Mouse Eggs

It is well documented that protein kinase A (PKA) acts as a negative regulator of M phase promoting factor (MPF) by phosphorylating cell division cycle 25 homolog B (Cdc25B) in mammals. However, the molecular mechanism remains unclear. In this study, we identified PKA phosphorylation sites in vitro by LC-MS/MS analysis, including Ser149, Ser229, and Ser321 of Cdc25B, and explored the role of Ser149 in G2/M transition of fertilized mouse eggs. The results showed that the overexpressed Cdc25B-S149A mutant initiated efficient MPF activation by direct dephosphorylation of Cdc2-Tyr15, resulting in triggering mitosis prior to Cdc25B-WT. Conversely, overexpression of the phosphomimic Cdc25B-S149D mutant showed no significant difference in comparison with the control groups. Furthermore, we found that Cdc25B-Ser149 was phosphorylated at G1 and S phases, whereas dephosphorylated at G2 and M phases, and the phosphorylation of Cdc25B-Ser149 was modulated by PKA in vivo. In addition, we examined endogenous and exogenous Cdc25B, which were expressed mostly in the cytoplasm at the G1 and S phases and translocated to the nucleus at the G2 phase. Collectively, our findings provide evidence that Ser149 may be another potential PKA phosphorylation target of Cdc25B in G2/M transition of fertilized mouse eggs and Cdc25B as a direct downstream substrate of PKA in mammals, which plays important roles in the regulation of early development of mouse embryos.

Serum interspecies differences in metabolic pathways of bradykinin and [des-Arg9]BK: influence of enalaprilat

1996 ◽  
Vol 271 (4) ◽  
pp. H1340-H1347 ◽  
Author(s):  
A. Decarie ◽  
P. Raymond ◽  
N. Gervais ◽  
R. Couture ◽  
A. Adam
Keyword(s):  
Metabolic Pathways ◽  
Neutral Endopeptidase ◽  
Thrombin Inhibitor ◽  
Rat Serum ◽  
Highly Sensitive ◽  
Significant Difference ◽  
Hydrolysis Of

Among the different enzymes responsible for the metabolism of bradykinin (BK), three peptidases look relevant in vivo: kininase I (KI), which transforms BK into its active metabolite, [des-Arg9]BK; kininase II (KII); and neutral endopeptidase, which inactivate BK and [des-Arg9]BK. The in vitro incubation of BK and [des-Arg9]BK in the serum of four species with or without enalaprilat and the quantification of the immunoreactivity of both peptides at different time intervals allowed the measurement of the kinetic parameters characterizing their metabolic pathways. Highly sensitive chemiluminescent enzyme immunoassays were used to measure the residual concentrations of BK and [des-Arg9]BK. Half-life (t1/2) of BK showed significant difference among species: rats (10 +/- 1 s) = dogs (13 +/- 1 s) < rabbits (31 +/- 1 s) < humans (49 +/- 2 s). t1/2 values of [des-Arg9]BK were also species dependent: rats (96 +/- 6 s) < < rabbits (314 +/- 6 s) = dogs (323 +/- 11 s) = humans (325 +/- 12 s). Enalaprilat significantly prevented the rapid BK and [des-Arg9]BK degradation in all species except that of [des-Arg9]BK in rat serum. Relative amount of BK hydrolyzed by serum KII was given as follows: rabbits (93.7 +/- 14.8%) = rats (83.6 +/- 6.7%) = humans (76.0 +/- 7.5%) > dogs (50.0 +/- 3.9%). Its importance in the hydrolysis of [des-Arg9]BK was 5.2 +/- 0.5% in rats < < 33.9 +/- 1.5% in humans < 52.0 +/- 1.1% in rabbits < 65.1 +/- 3.4% in dogs. The participation of serum KI in the transformation of BK into [des-Arg9]BK was dogs (67.2 +/- 5.3%) > > humans (3.4 +/- 1.2%) = rabbits (1.8 +/- 0.2%) = rats (1.4 +/- 0.3%). Finally, no significant difference on t1/2 values for BK and [des-Arg9]BK could be demonstrated between serum and plasma treated with either sodium citrate or a thrombin inhibitor. These results revealed striking species differences in the serum metabolism of kinins that could address at least partially some of the controversial data related to the cardioprotective role of kinins.

scholarly journals Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C

1996 ◽  
Vol 314 (3) ◽  
pp. 937-942 ◽  
Author(s):  
Karen L. CRAIG ◽  
Calvin B. HARLEY
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Cos Cells ◽  
Phospholipid Hydrolysis ◽  
Kinase C

During platelet activation, receptor-coupled phospholipid hydrolysis stimulates protein kinase C (PKC) and results in the phosphorylation of several proteins, the most prominent being pleckstrin. Pleckstrin is composed of two repeated domains, now called pleckstrin homology (PH) domains, separated by a spacer region that contains several consensus PKC phosphorylation sites. To determine the role of PKC-dependent phosphorylation in pleckstrin function, we mapped the phosphorylation sites in vivo of wild-type and site-directed mutants of pleckstrin expressed in COS cells. Phosphorylation was found to occur almost exclusively on Ser-113 and Ser-117 within the sequence 108-KFARKS*TRRS*IRL-120. Phosphorylation of these sites was confirmed by phosphorylation of the corresponding wild-type and mutant synthetic peptides in vitro.

Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin

Development ◽  
1993 ◽  
Vol 119 (4) ◽  
pp. 1079-1091 ◽  
Author(s):  
E.L. George ◽  
E.N. Georges-Labouesse ◽  
R.S. Patel-King ◽  
H. Rayburn ◽  
R.O. Hynes
Keyword(s):  
Mutant Allele ◽  
Mouse Embryos ◽  
Neural Tubes ◽  
Early Embryonic Lethality ◽  
Homozygous Mutant ◽  
Anterior Posterior ◽  
Embryonic Vessels

To examine the role of fibronectin in vivo, we have generated mice in which the fibronectin gene is inactivated. Heterozygotes have one half normal levels of plasma fibronectin, yet appear normal. When homozygous, the mutant allele causes early embryonic lethality, proving that fibronectin is required for embryogenesis. However, homozygous mutant embryos implant and initiate gastrulation normally including extensive mesodermal movement. Neural folds also form but the mutant embryos subsequently display shortened anterior-posterior axes, deformed neural tubes and severe defects in mesodermally derived tissues. Notochord and somites are absent; the heart and embryonic vessels are variable and deformed, and the yolk sac, extraembryonic vasculature and amnion are also defective. These abnormalities can be interpreted as arising from fundamental deficits in mesodermal migration, adhesion, proliferation or differentiation as a result of the absence of fibronectin. The nature of these embryonic defects leads to reevaluation of suggested roles for fibronectin during early development based on results obtained in vitro and in embryos of other species.

An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2333-2333
Author(s):  
Brian D. Adams ◽  
Shangqin Guo ◽  
Haitao Bai ◽  
Changchun Xiao ◽  
E. Premkumar Reddy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

Insight in Protein S Deficiency From Mouse Models

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 529-529
Author(s):  
Sara Calzavarini ◽  
François Saller ◽  
Jose A. Fernandez ◽  
Linda Kadi ◽  
Anne C. Brisset ◽  
...  
Keyword(s):  
Mouse Models ◽  
Purpura Fulminans ◽  
Protein S ◽  
Negative Regulator ◽  
Thrombotic Risk ◽  
Increased Risk ◽  
Deficient Mice

Abstract Abstract 529 Protein S (ProS) is an important negative regulator of blood coagulation. Its physiological importance is evident in purpura fulminans and other life-threatening thrombotic disorders typical of ProS deficient patients. Our previous characterization of ProS deficiency in mouse models has shown similarities with the human phenotypes: heterozygous ProS-deficient mice (Pros+/−) had increased thrombotic risk whereas homozygous deficiency in ProS (Pros−/−) was incompatible with life (Blood 2009; 114:2307-2314). In tissues, ProS exerts cellular functions by binding to and activating tyrosine kinase receptors of the Tyro3 family (TAM) on the cell surface. To extend the analysis of coagulation defects beyond the Pros−/− phenotype and add new insights into the sites of synthesis ProS and its action, we generated mice with inactivated ProS in hepatocytes (Proslox/loxAlbCre+) as well as in endothelial and hematopoietic cells (Proslox/loxTie2Cre+). Both models resulted in significant reduction of circulating ProS levels and in a remarkable increased thrombotic risk in vivo. In a model of tissue factor (TF)-induced venous thromboembolism (VTE), only 17% of Proslox/loxAlbCre+ mice (n=12) and only 13% of Proslox/loxTie2Cre+ mice (n=14) survived, compared with 86% of Proslox/lox mice (n=14; P<0.001). To mimic a severe acquired ProS deficiency, ProS gene was inactivated at the adult stage using the polyI:C-inducible Mx1-Cre system (Proslox/loxMx1Cre+). Ten days after polyI:C treatment, Proslox/loxMx1Cre+ mice developed disseminated intravascular coagulation with extensive lung and liver thrombosis. It is worth noting that no skin lesions compatible with purpura fulminans were observed in any of the above-described models of partial ProS deficiency. In order to shed light on the pathogenesis of purpura fulminans, we exposed the different ProS-deficient mice to warfarin (0.2 mg/day). We observed that Pros+/−, Proslox/loxAlbCre+ and Proslox/loxTie2Cre+ mice developed retiform purpura (characterized by erythematous and necrotic lesions of the genital region and extremities) and died after 3 to 5 days after the first warfarin administration. In human, ProS is also synthesized by megakaryocytes and hence stored at high concentrations in circulating platelets (pProS). The role of pProS has been investigated by generating megakaryocyte ProS-deficient model using the PF4 promoter as Cre driver (Proslox/loxPf4Cre+). In the TF-induced VTE model, Proslox/loxPf4Cre+ (n=15) mice showed a significant increased risk of thrombosis compared to Proslox/lox controls (n=14; survival rate 47% and 86%, respectively; P<0.05). Furthermore, preliminary results suggest survival to be associated with higher circulating ProS levels. In order to evaluate the potential role of pProS in thrombus formation, we investigated the thrombotic response to intravenous injection of collagen-epinephrine in vivo and platelet function in vitro. Both in vivo and in vitro experiments showed similar results between Proslox/loxPf4Cre+ and Proslox/lox, indicating that platelet reactivity was not influenced by the absence of pProS. These data suggest that pProS is delivered at the site of thrombosis to inhibit thrombin generation. We further investigated the ability of ProS to function as a ligand of TAM receptors, by using homozygous and heterozygous deficient mice for both the TAM ligands ProS and Gas6. Gas6−/−Pros−/− mice died in utero and showed comparable dramatic bleeding and thrombotic phenotype as described for Pros−/− embryos. In conclusion, like complete ProS deficiency, double deficiency in ProS and Gas6 was lethal, whereas partial ProS deficiency was not. Mice partially deficient in ProS displayed a prothrombotic phenotype, including those with only deficiency in pProS. Purpura fulminans did not occur spontaneously in mice with partial Pros deficiency but developed upon warfarin administration. Thus, the use of different mice models of ProS deficiency can be instrumental in the study of its highly variable thrombotic phenotype and in the investigation of additional roles of ProS in inflammation and autoimmunity through TAM signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Latex and nonlatex orthodontic elastics: In vitro and in vivo evaluations of tissue compatibility and surface structure

2015 ◽  
Vol 86 (2) ◽  
pp. 278-284 ◽  
Author(s):  
Sara Martínez-Colomer ◽  
Patrícia Gaton-Hernández ◽  
Fábio Lourenço Romano ◽  
Andiara De Rossi ◽  
Sandra Yasuyo Fukada ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Surface Structure ◽  
Null Hypothesis ◽  
Microscopic Analysis ◽  
Plasma Extravasation ◽  
Control Groups ◽  
Tissue Compatibility ◽  
Significant Difference

ABSTRACT Objective:  To test the null hypothesis that there is no difference between latex and nonlatex orthodontic elastics with respect to tissue compatibility and surface structure. Materials and Methods:  Latex and nonlatex elastics were implanted in the subcutaneous connective tissue of 45 Wistar rats. In the control groups, no material was implanted (sham). After 24 hours, 3, 7, 14, and 21 days, the animals were euthanized; tissue samples were processed and analyzed by descriptive and semi-quantitative microscopic analysis and quantification of plasma extravasation. The surface structure of elastics was evaluated by scanning electron microscopy (SEM). The results were analyzed by analysis of variance (ANOVA), Tukey test and Kruskal-Wallis test at 5% significance level. Results:  Peri-implant plasma extravasation was significantly higher (P &lt; .05) in the animals that received latex elastics compared with those with nonlatex elastics and those that were control animals. The microscopic analysis revealed a more intense inflammatory infiltrate in the initial periods without statistically significant difference (P &gt; .05) between the experimental and control groups. The SEM analysis revealed that the latex elastics presented microspheres and porosities, while the nonlatex elastics exhibited crystals on their surface and absence of porosities. Conclusion:  The null hypothesis was rejected since the latex elastics were more irritating to the connective tissue than the nonlatex elastics in the initial periods and presented a more porous surface.

A simplified biopsy method for precompacted mouse embryos: A technical report

2002 ◽  
Vol 50 (4) ◽  
pp. 469-479 ◽  
Author(s):  
S. Bodó ◽  
L. Laczkó ◽  
Gabriella Horváth ◽  
Keyword(s):  
Genetic Diagnosis ◽  
In Utero ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Embryo Biopsy ◽  
Control Groups ◽  
Technical Report ◽  
Significant Difference ◽  
Biopsy Method

This article presents a new, simple and rapid embryo biopsy method. The blastomere for genetic analysis can be separated from a precompacted mouse embryo after a partial zona digestion with the use of a holding pipette. For the micromanipulation only two microcapillaries and micromanipulators are needed. The development of the biopsied embryos was studied during in vitro culture and in utero following embryo transfer. There was no significant difference between the treated and the control groups in the ratio of embryos that developed to the blastocyst stage, although the biopsied embryos were delayed in their development because they contained significantly fewer cells compared to the control ones at the same stage. Although there was no difference in the ratio of implantation, the development of the biopsied embryos in utero was also delayed 12-24 hours on the 9th day of pregnancy. No difference in development was visible from the 13th day of pregnancy. Statistically, no differences were found in the developmental ratio (number of developed fetuses/transferred embryos) of the control and treated embryos during gastrulation (9th day of pregnancy), at the beginning of organogenesis (13th day of pregnancy) and before birth (19th day of pregnancy). The embryo biopsy method presented here can be a new and useful tool for preimplantation genetic diagnosis.

Anthelmintic activity of some Mediterranean browse plants against parasitic nematodes

Parasitology ◽  
2009 ◽  
Vol 137 (4) ◽  
pp. 685-696 ◽  
Author(s):  
F. MANOLARAKI ◽  
S. SOTIRAKI ◽  
A. STEFANAKIS ◽  
V. SKAMPARDONIS ◽  
M. VOLANIS ◽  
...  
Keyword(s):  
Anthelmintic Activity ◽  
Experimental Period ◽  
Significant Inhibition ◽  
Parasitic Nematodes ◽  
Control Groups ◽  
Mediterranean Plants ◽  
Cell Volumes

SUMMARYThe anthelmintic properties of tannin-rich plants are being explored as an alternative to chemical drugs. Most data have been acquired on legume forages, but only few on browse plants. The present study aimed to (i) screen the in vitro effects of extracts from 7 Mediterranean plants on Haemonchus contortus, (ii) verify the role of tannins using an inhibitor, polyvinyl polypyrrolidone (PVPP) and (iii) verify the in vivo effects of extracts from 4 plants. Significant inhibition was shown in vitro using a larval migration inhibition (LMI) assay for all extracts except that from Olea europaea var. koroneiki. After adding PVPP, the LMI values were restored to control levels for all plants except Pistacia lentiscus and Ceratonia siliqua, confirming a role for tannins in the activity. In the in vivo experiment, 48 lambs composed 6 groups, depending on diet. On Day 0, groups G1–G5 received H. contortus and Trichostrongylus colubriformis larvae and G6 remained uninfected. The various diets were distributed from Days 14 to 45: P. lentiscus (G1), Quercus coccifera (G2), C. siliqua (G3), Onobrychis viciifolia (G4), or Medicago sativa for the 2 control groups (G5, G6). Egg excretion, packed cell volumes (PCVs) and inorganic phosphate were measured weekly throughout the entire experimental period. At slaughter, the worms were enumerated and their fecundity assessed. Consumption of the 4 browser plants did not provoke differences in pathophysiological measurements but there were significant decreases in egg excretion, mainly explained by significant decreases in worm fecundity for both species, without any statistical difference in worm numbers.

scholarly journals Inhibition of KIF14 Suppresses Tumor Cell Growth and Promotes Apoptosis in Human Glioblastoma

2015 ◽  
Vol 37 (5) ◽  
pp. 1659-1670 ◽  
Author(s):  
Wei Huang ◽  
JunYu Wang ◽  
Danfeng Zhang ◽  
Wen Chen ◽  
Lijun Hou ◽  
...  
Keyword(s):  
Normal Brain ◽  
Ki 67 ◽  
Akt Phosphorylation ◽  
M Phase ◽  
Underlying Mechanisms ◽  
Sirna Knockdown ◽  
Kinesin Superfamily

Background/Aims: The mitotic kinesin superfamily protein KIF14 is essential for cytokinesis and chromosome segregation, and increased KIF14 expression is related to a variety of human cancers. However, the role of KIF14 in the development and malignant progression of astrocytomas and the underlying mechanisms remain unclear. The present study examined the relation between KIF14 and the pathogenesis of malignant astrocytoma. Methods and Results: The role of KIF14 in astrocytoma development and progression was investigated by analyzing KIF14 expression using SYBR Green quantitative real-time RT-PCR, western blotting and immunohistochemistry in human astrocytoma and normal brain tissues. KIF14 expression was higher in astrocytoma samples, and was positively correlated with pathological grade and proliferative activity indicated by Ki-67 staining. SiRNA knockdown of KIF14 inhibited tumor growth in vitro and in vivo, attenuated anchorage-independent growth, and induced G2/M phase arrest, cytokinesis failure and apoptosis in glioblastoma cell lines in association with decreased AKT phosphorylation and activity. Conclusions: The upregulation of KIF14 in astrocytoma is associated with disease severity, and suppression of KIF14 inhibits cell proliferation and induces apoptosis through a mechanism involving the inactivation of AKT signaling, suggesting that KIF14 plays an important role in astrocytoma tumorigenesis and could be a promising molecular target for anticancer therapy.

scholarly journals Molecular Determinants for PspA-Mediated Repression of the AAA Transcriptional Activator PspF

2005 ◽  
Vol 187 (9) ◽  
pp. 3238-3248 ◽  
Author(s):  
Sarah Elderkin ◽  
Patricia Bordes ◽  
Susan Jones ◽  
Mathieu Rappas ◽  
Martin Buck
Keyword(s):  
Regulatory Protein ◽  
Membrane Integrity ◽  
Transcriptional Activator ◽  
Negative Regulator ◽  
E Coli ◽  
Molecular Determinants ◽  
Negative Effect

ABSTRACT The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the σ54 subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of σ54 AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal α-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

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