Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Differential activation of two protease-activated protein kinases from reticulocytes by a Ca2+-stimulated protease and identification of phosphorylated translational components

Two protein kinases have been partially purified from rabbit reticulocytes and shown to be activated by limited proteolysis with trypsin [S.M. Tahara and J.A. Traugh (1981) J. Biol. Chem. 256, 11558-11564; P.T. Tuazon, W.C. Merrick, and J.A. Traugh (1980) J. Biol. Chem. 255, 10954-10958]. Reticulocyte lysate was examined for protease activities which might be involved in activation of the protein kinases in vivo. Two neutral proteases, differentially activated by Fe2+ and Ca2+, were identified and partially purified. The Ca2+-stimulated protease specifically activated protease-activated kinase II; no effect was observed on protease-activated kinase I. The Fe2+-stimulated protease was not active on either protein kinase. The protease-activated kinases were examined using initiation factors (eIF) and 40-S ribosomal subunits as substrate. Protease-activated kinase I phosphorylated one subunit of eIF-3 (Mr 130000), eIF-4B and 40-S ribosomal protein S10. Protease-activated kinase II modified the beta subunit of eIF-2 (Mr 53000) and 40-S ribosomal protein S6. The substrate specificities are unique when compared with other cAMP-dependent and cAMP-independent protein kinases from reticulocytes.     

Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141

Pak2, a member of the p21-activated protein kinase (Pak) family, is activated in response to a variety of stresses and is directly involved in the induction of cytostasis. At the molecular level Pak2 binds Cdc42(GTP), translocating Pak2 to the endoplasmic reticulum where it is autophosphorylated and activated. Pak2 is autophosphorylated at eight sites; Ser-141 and Ser-165 in the regulatory domain and Thr-402 in the activation loop are identified as key sites in activation of the protein kinase. The function of phosphorylation of Ser-141 and Ser-165 on the activation was analyzed with wild-type (WT) and mutants of Pak2. With S141A, the level of autophosphorylation was reduced to 65% as compared with that of WT and S141D with a concomitant 45% reduction in substrate phosphorylation, indicating that phosphorylation at Ser-141 is required for optimal activity. Autophosphorylation inhibited the interaction between WT Pak2 and Cdc42(GTP). In 293T cells, WT Pak2, S141A, and S141D formed a stable complex with the constitutively active mutant Cdc42 L61, but not with the dominant negative Cdc42 N17. As shown in glutathione S-transferase pull-down assays, S141A bound to Cdc42(GTP) at a 6-fold higher level than that of S141D. In contrast, the S165A and S165D mutants had no effect on autophosphorylation, binding to Cdc42, or activation of Pak2. In summary, autophosphorylation of Ser-141 was required for activation of Pak2 and down-regulated the interaction of Pak2 with Cdc42. A model is proposed suggesting that binding of Cdc42 localizes Pak2 to the endoplasmic reticulum, where autophosphorylation alters association of the two proteins.     

p21-activated protein kinase gamma-PAK is translocated and activated in response to hyperosmolarity. Implication of Cdc42 and phosphoinositide 3-kinase in a two-step mechanism for gamma-PAK activation

A member of the family of p21-activated protein kinases, gamma-PAK, has cytostatic properties and is activated during apoptosis and in response to DNA damage. To determine whether gamma-PAK is activated by other types of cell stress and to assess its mechanism of activation, the response of gamma-PAK to hyperosmotic stress was examined. In 3T3-L1 mouse fibroblasts, there are two pools of gamma-PAK: the majority of the protein kinase is soluble and has low specific activity, whereas gamma-PAK associated with the particulate fraction has significantly higher specific activity. Hyperosmolarity promotes translocation of gamma-PAK from the soluble to the particulate fraction; this parallels activation of the protein kinase. Activation but not translocation of gamma-PAK is wortmannin-sensitive, suggesting the involvement of a phosphoinositide 3-kinase-related activity. gamma-PAK translocation in response to hyperosmolarity parallels Cdc42 translocation to the particulate fraction in vivo and can be induced in vitro by guanosine 5'-3-O-(thio)triphosphate. Cotransfection of gamma-PAK with constitutively active Cdc42 induces gamma-PAK activation and translocation, whereas inactive Cdc42 inhibits both processes in response to hyperosmotic stress, suggesting that Cdc42 has a role in the translocation and activation of gamma-PAK. alpha-PAK is not activated in response to hyperosmolarity in 3T3-L1 cells. A two-step model of gamma-PAK activation is presented.     

Thesaurus-based disambiguation of gene symbols

Background  

Massive text mining of the biological literature holds great promise of relating disparate information and discovering new knowledge. However, disambiguation of gene symbols is a major bottleneck.     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Behavioural and physiological consequences of acute social defeat in growing gilts: effects of the social environment

Endocrine, behavioural and immunologic processes, together with body growth, were evaluated in gilts that were defeated at 10 weeks of age in resident-intruder tests. Immediately after defeat, gilts were either separated from or reunited with a familiar conspecific (litter-mate; always a barrow). Gilts were assigned to one of four treatments: (a) DI: defeat, followed by isolation (separation from original litter-mate; n=8); (b) I: no defeat, isolation (control group; n=9); (c) DP; defeat, followed by pair-housing (reunion with original litter-mate; n=8); and (d) P: no defeat, pair-housing (control group; n=8). The following general conclusions were derived: (1) social defeat caused pronounced short-term elevations in hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal medullary activities, and of prolactin levels. Moreover, as soon as 1h after defeat, percentages of blood lymphocytes and neutrophilic granulocytes were, respectively, decreased and increased; (2) social defeat had some long-lasting influence on behaviour and physiology, but isolation predominantly determined responses in the longer term. Defeat, as well as isolation, resulted in increased cardiovascular activities compared to P controls, as observed in a novel object test (NOT: +7 days) and an aversion test (AVT: +14 days). Moreover, defeated as well as isolated gilts did not habituate to a repeated novel environment test (NET: -7, +2 and +7 days) in terms of frequencies of vocalising, whereas P controls did. Isolation, through the separation from any other pig, was responsible for the other observed long-term characteristics, which developed progressively. Isolated gilts showed high mobilities and high cortisol responses in the repeated NET (+7 days), not being habituated. This contrasted the reactions of pair-housed gilts, which were much reduced. In addition to their high cardiovascular activities in the NOT and the AVT, isolated gilts also displayed higher heart rates in the repeated NET and during human presence following the NOT, compared to pair-housed gilts. Finally, isolated gilts were more inhibited to approach a novel object (in the NOT) than pair-housed pigs; and (3) stress responses of defeated gilts were modulated by the subsequent social environment. Stimulation of the HPA-axis (plasma- and salivary cortisol) was prolonged in those defeated gilts which were isolated (observed in the first hour). Changes in leucocyte subsets were still observed after 3 days in DI, but were 'normalised' within 1 day in DP gilts. Two days after defeat, habituation to the repeated NET in terms of mobility and salivary cortisol responses occurred in control and DP gilts, but not in DI gilts. We argue that these effects of the social environment shortly after defeat were related to a stress-reducing effect of a stable social relationship, i.e. social support.     

Phosphorylation of human p53 on Thr-55

The pleiotropic function of p53 is believed to be greatly influenced by phosphorylation, and several sites on p53 are known to be targets for distinct protein kinases. In this study, we observed that affinity-purified p53 from overexpressing cells was phosphorylated by a co-purified protein kinase in vitro. To identify phosphorylation site(s), the resulting phosphorylated p53 protein was subjected to primary and secondary proteolytic cleavage, and phosphopeptides were fractionated by a two-dimensional peptide gel system. Phosphoamino acid analysis and manual Edman degradation of the isolated phosphopeptides enabled us to unequivocally identify Thr-55 as the major phosphorylation site on p53. Furthermore, comparative phosphopeptide mapping data suggest that DNA-PK is not the kinase responsible for this phosphorylation. Significantly, using a phospho-specific antibody for Thr-55, we have shown that Thr-55 is indeed phosphorylated in vivo. These data define Thr-55 as a novel phosphorylation site and for the first time show threonine phosphorylation of human p53.