Luteinizing hormone receptor activation in ovarian granulosa cells promotes protein kinase A-dependent dephosphorylation of microtubule-associated protein 2D

The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.     

Personalized Method for Interpreting Dreams (PMID)—As Applied to Relationship Issues

The purpose of this study was to develop a dreamwork model that would help individuals deal with relationship issues. Seventy dreams, involving seven major relationships, were selected from the woman participant's dreams. A dream interpretation model, the Personalized Method for Interpreting Dreams (PMID) was developed. Well-founded concepts in the PMID are: 1) dreams reflect emotions; and, 2) pre-dream thoughts, current circumstances, and personal definitions build dream meanings. The newest dreamwork concept of the PMID is the systemic perspective that relationship issues are best understood by discovering how relationship experiences influence our thoughts, emotions and behavior in other relationships. With a dreamwork systemic approach, the individual gathers together and studies series of dreams about major relationships in his or her life, primarily the family. Results of the thesis study show that the participant's use of the model was a factor in reducing stressful relationship issues.     

Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer

Background  

We have used commercially available cDNA arrays to identify EphB4 as a gene that is up-regulated in colon cancer tissue when compared with matched normal tissue from the same patient.     

Influence of light on feeding anticipatory activity in goldfish

Most animals when presented with food on a periodic basis develop feeding anticipatory activity (FAA). The objective of the present study was to investigate the synchronization of activity rhythms to light and feeding in single goldfish given a single daily meal and exposed to different light regimes. In the 1st experiment, the group of fish fed at the end of the day showed a longer FAA (228 +/- 27 min) than the group fed 1 h after lights-on (97 +/- 31 min). In the 2nd experiment, goldfish under conflicting zeitgebers, LD (T = 26) and feeding (T = 24) cycles, initially synchronized to the light cycle although feeding time gradually assumed greater importance as the experiment progressed. In the 3rd experiment, after altering the feeding cycles by advancing or delaying feeding time by 4 h in LD 0.25:23.75, most goldfish resynchronized to the feeding stimulus and developed FAA. In conclusion, the authors' results revealed that FAA in goldfish is driven by an endogenous timing system that is entrainable by periodic feeding and influenced by the light regime.     

Altered directionality in the Cre-LoxP site-specific recombination pathway

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.     

Effects of (+)-totarol, a diterpenoid antibacterial agent, on phospholipid model membranes

(+)-Totarol, a highly hydrophobic diterpenoid isolated from Podocarpus spp., is inhibitory towards the growth of diverse bacterial species. (+)-Totarol decreased the onset temperature of the gel to liquid-crystalline phase transition of DMPC and DMPG membranes and was immiscible with these lipids in the fluid phase at concentrations greater than 5 mol%. Different (+)-totarol/phospholipid mixtures having different stoichiometries appear to coexist with the pure phospholipid in the fluid phase. At concentrations greater than 15 mol% (+)-totarol completely suppressed the gel to liquid-crystalline phase transition in both DMPC and DMPG vesicles. Incorporation of increasing amounts of (+)-totarol into DEPE vesicles induced the appearance of the H(II) hexagonal phase at low temperatures in accordance with NMR data. At (+)-totarol concentrations between 5 and 35 mol% complex thermograms were observed, with new immiscible phases appearing at temperatures below the main transition of DEPE. Steady-state fluorescence anisotropy measurements showed that (+)-totarol decreased and increased the structural order of the phospholipid bilayer below and above the main gel to liquid-crystalline phase transition of DMPC respectively. The changes that (+)-totarol promotes in the physical properties of model membranes, compromising the functional integrity of the cell membrane, could explain its antibacterial effects.     

Modulation of polymorphic properties of dielaidoylphosphatidylethanolamine by the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine

The capacity of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH3) to modulate the polymorphic properties of dielaidoylphosphatidylethanolamine has been studied using biophysical techniques. Differential scanning calorimetry showed that ET-18-OCH3 depresses the onset of the Lbeta to Lalpha phase transition, decreasing also DeltaH of the transition. At the same time, the onset of the transition from Lalpha to inverted hexagonal HII phase was gradually increased as the ether lipid concentration was increased, totally disappearing at concentrations higher than 5 mol%. Small-angle X-ray diffraction and 31P-NMR confirmed that ET-18-OCH3 induced that the appearance of the inverted hexagonal HII phase was shifted towards higher temperatures completely disappearing at concentrations higher than 5 mol%. These results were used to elaborate a partial phase diagram and they were discussed as a function of the molecular action of ET-18-OCH3.     

Pre-M phase-promoting factor associates with annulate lamellae in Xenopus oocytes and egg extracts

We have used complementary biochemical and in vivo approaches to study the compartmentalization of M phase-promoting factor (MPF) in prophase Xenopus eggs and oocytes. We first examined the distribution of MPF (Cdc2/CyclinB2) and membranous organelles in high-speed extracts of Xenopus eggs made during mitotic prophase. These extracts were found to lack mitochondria, Golgi membranes, and most endoplasmic reticulum (ER) but to contain the bulk of the pre-MPF pool. This pre-MPF could be pelleted by further centrifugation along with components necessary to activate it. On activation, Cdc2/CyclinB2 moved into the soluble fraction. Electron microscopy and Western blot analysis showed that the pre-MPF pellet contained a specific ER subdomain comprising "annulate lamellae" (AL): stacked ER membranes highly enriched in nuclear pores. Colocalization of pre-MPF with AL was demonstrated by anti-CyclinB2 immunofluorescence in prophase oocytes, in which AL are positioned close to the vegetal surface. Green fluorescent protein-CyclinB2 expressed in oocytes also localized at AL. These data suggest that inactive MPF associates with nuclear envelope components just before activation. This association may explain why nuclei and centrosomes stimulate MPF activation and provide a mechanism for targeting of MPF to some of its key substrates.