Deciduogenic effects of mediators of the polyphosphatidylinositol pathway in pseudopregnant mice

Intraluminal injections (15 l) of either concanavalin A (125 g) or ionophore A 23187 (0.01 mol) induced a decidual cell reaction (DCR) in the uterus of day 4.5 pseudopregnant mice. However, when these agents were administered in different combinations with each other or with CaCl₂ (15 mol) and phorbol-12-myristate-13-acetate (1.6 nmol), interacting effects occurred to either enhance or inhibit each of the others' independent deciduogenic capacities. The results suggest that the polyphosphatidylinositol pathway and Ca²⁺ are involved in the induction of the DCR in mice with complex interactions occurring between the active components of the pathway to modulate the outcome of the transformation process.     

Changes in plasma levels of metabolites during long-term fasting in the goose]

In fed geese, plasma levels of glucose and alanine were 1.9 g.l-1 and 560 mumol.l-1, respectively. During a long fast (40 days), plasma glucose and alanine were maintained at a high level (1.5-1.8 g.l-1 and 370-540 mumol.l-1, respectively). Plasma level of acetoacetate was very low (40 mumol.l-1); by contrast, plasma level of beta hydroxybutyrate reached very high values (20 mmol.l-1) after about 20 days of fasting, then it decreased. Plasma levels of lactate and pyruvate decreased along the course of the fast, from 2 500 to 2 000 mumol.l-1 and 220 to 170 mumol.l-1, respectively.     

Differential stimulation of signaling pathways initiated by Edg-2 in response to lysophosphatidic acid or sphingosine-1-phosphate

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are produced during cell activation and have multiple effects on cells. A family of seven transmembrane-spanning domain G-protein-coupled receptors, named Edg, mediate these effects of LPA and S1P. In this study, transient overexpression of Edg-2 sensitized MG63 human osteosarcoma cells to both LPA- and S1P-mediated stimulation of fibronectin matrix deposition and actin stress fiber formation. Both lipids were active in the 1-20 nM concentration range on cells transfected with Edg-2 as compared to the 10-200 nM range on mock-transfected cells. The signaling pathway for matrix deposition by Edg-2-transfected cells was Rho dependent. Overexpression of Edg-2 also caused a tenfold decrease in the concentration of either LPA or S1P that activated MAPKinase (Erkl/2) in MG63 cells. LPA- or S1P-stimulated activation of Erkl/2 was Gi dependent. These results indicate that, in MG63 cells, Edg-2 mediates actin stress fiber formation, fibronectin matrix assembly, and MAPKinase activation in response to either LPA or S1P.     

Decidual cell reaction in ovariectomized-adrenalectomized rats

Enhancement of decidual cell reaction (DCR) following adrenalectomy was reversed by corticosterone as well as indomethacin. The results suggest the adrenal involvement in DCR through uterine prostaglandin production.     

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.     

Circadian rhythmicity in phosphorylase activity and glycogen content in the heart muscle of the scorpion,Heterometrus fulvipes,C.L. Koch

Summary Maximal activity levels of phosphorylase A and AB at 20.00 h alternate with minimal levels at 08.00 h of the day, while the glycogen content exhibited a reverse trend in the heart of the scorpion,Heterometrus fulvipes.Acknowledgment. The authors thank Prof. K.S. Swami, for providing facilities. The financial assistance rendered by UGC (to VJ) and ICMR (to DCR) is gratefully acknowledged.     

Ureido-ethyl-imidazolines active against autochthonous diethylnitrosamine-induced epidermoid,papillary and adenocarcinomatous tumors of the respiratory tract of Syrian hamsters and against human bronchogenic carcinomas in nu/nu mice

Summary The detection of a new class of tumor inhibiting substances is described. Employing a chemical reaction discovered several years ago, a series of imidazolinylureas were prepared. It was found that some compounds of this group were active against diethylnitrosamine (DENA)-induced tumours in hamsters. CGP 15 720 A (1-{2-[2-(4-pyridyl)-2-imidazoline-l-yl]-ethyl}-3-(4-carboxy-phenyl)urea,Xb), the most active compound at present, was developed through a series of structural variations. CGP 15 720 A inhibits significantly in oral or parenteral treatment with well tolerated doses (10–30 mg/kg) the progressive growth of autochthonous, DENA-induced papillary, epidermoid and adenocarcinomatous tumors of the respiratory system in Syrian hamsters and prolongs significantly the survival. The substance also inhibits significantly the growth of 2 poorly differentiated human epidermoid or anaplastic bronchogenic carcinomas in nu/nu Balb/c mice and prolongs the mean survival time. In these mice, the substance is also active against the rodent ascites tumors Ehrlich carcinoma, CrSa 180 and Yoshida Sa AH 66, although it is only marginally active or inactive against these tumors in normal mice or rats. — In the therapeutic trials, hamsters tolerated the highest dose administered for 4 weeks, 1000 mg/kg p.o., without signs or symptoms of toxicity.Editorial remarks. There is still an urgent medical need for effective and welltolerated drugs for the treatment of the most common forms of cancer, such as bronchial carcinoma, or for post-operative prophylaxis against relapse and metastasis. — The old-established screening method based on rapidly proliferating acute transplantable lymphatic leukemias in the mouse that is applied in the major cancer research centers has certainly achieved some measure of clinical success, inasmuch as the mean duration of survival of patients with acute lymphatic leukemia has increased from 3 months to about 6 years and similar activity has been found in some rapidly proliferating lymphomas, sarcomas and teratomas.The authors were convinced, however, that chemotherapeutic agents effective against lung cancer could only be found with the help of new specific animal models. They developed a model of an autochthonous tumor in the hamster, applied it in extensive series of experiments, and succeeded in synthesizing and identifying a group of compounds that were both effective and well tolerated. They describe the synthesis and biological activity of CGP 15 720, the compound with the highest therapeutic index and an apparently non-cytotoxic mode of action.     

Intracellular sterol trafficking

Summary Sterols are acquired by cells either biosynthetically by the interaction of cytoplasmic and endoplasmic reticulum elements, or by endocytosis. The subcellular distribution of sterols, however, argues that sterols are trafficked quickly from sites of acquisition to target membranes, particularly the plasma membrane. The mechanisms mediating this movement might include aqueous diffusion, vesicles of either a unique pathway or of the protein secretory pathway, or carrier proteins. These mechanisms are discussed and the limited data concerning each are presented. Finally, a theory is proposed which describes how sterols and other membrane reinforcing molecules might have driven the evolution of intracellular membranes, thus establishing the dynamic membrane system of modern eukaryotes.     

Intracellular sterol trafficking

Sterols are acquired by cells either biosynthetically by the interaction of cytoplasmic and endoplasmic reticulum elements, or by endocytosis. The subcellular distribution of sterols, however, argues that sterols are trafficked quickly from sites of acquisition to target membranes, particularly the plasma membrane. The mechanisms mediating this movement might include aqueous diffusion, vesicles of either a unique pathway or of the protein secretory pathway, or carrier proteins. These mechanisms are discussed and the limited data concerning each are presented. Finally, a theory is proposed which describes how sterols and other membrane reinforcing molecules might have driven the evolution of intracellular membranes, thus establishing the dynamic membrane system of modern eukaryotes.     

Interaction of cortisol and epinephrine in the regulation of leucine kinetics in man

To assess the interaction of the two major stress hormones epinephrine and cortisol in the regulation of leucine kinetics in man, epinephrine (50 ng/kg/min) was infused either alone or in combination with cortisol (2 micrograms/kg/min) into two groups of 6 postabsorptive normal male subjects during 180 min. Plasma leucine concentrations decreased by 28% (p less than 0.05) from baseline during epinephrine treatment (plasma levels 515 pg/ml); this was due to a decrease of leucine appearance (determined by 1-13C-leucine infusions) by 23% (p less than 0.025); leucine oxidation decreased by 29% (p less than 0.05). However, when plasma cortisol concentrations were elevated to supraphysiological levels (16.3 mumol/l) during epinephrine administration, the decreases of leucine plasma concentrations, appearance and oxidation were abolished. Plasma glucose and FFA concentrations were similarly elevated during both kinds of treatment. Since leucine appearance represents a measurement of total body protein breakdown and leucine disappearance into non-oxidative pathways reflects protein synthesis, the data indicate that plasma epinephrine concentrations during severe stress exert a protein anabolic effect in man which may counteract catabolic properties of elevated plasma cortisol.     

Regulation of insulin receptor function

Resistance to the biological actions of insulin contributes to the development of type 2 diabetes and risk of cardiovascular disease. A reduced biological response to insulin by tissues results from an impairment in the cascade of phosphorylation events within cells that regulate the activity of enzymes comprising the insulin signaling pathway. In most models of insulin resistance, there is evidence that this decrement in insulin signaling begins with either the activation or substrate kinase activity of the insulin receptor (IR), which is the only component of the pathway that is unique to insulin action. Activation of the IR can be impaired by post-translational modifications of the protein involving serine phosphorylation, or by binding to inhibiting proteins such as PC-1 or members of the SOCS or Grb protein families. The impact of these processes on the conformational changes and phosphorylation events required for full signaling activity, as well as the role of these mechanisms in human disease, is reviewed in this article. Received 3 August 2006; received after revision 1 December 2006; accepted 8 January 2007     

Long-term depression of two primary immune responses induced by a single dose of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC)

Summary 2 primary immune responses (anti-SRBC antibody response and allograft rejection) have been tested in mice at various time intervals after single doses of either DTIC or cyclophosphamide. The DTIC-induced immunodepression proved to be extremely long-lasting, being still detectable after 2 months.This work was supported by Project NO1-CB 64054 of the U.S.-Italy Cooperative Science Program.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion.

The role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca2+, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca2+ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 mumol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 mumol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca2+. Increasing the Ca2+ concentration to 1.26 mmol/l in TCM 199 during the 22-24 h exposure to glucose (16.7 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca2+ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Transgenic and knock-out mice for deciphering the roles of EGFR ligands

Generation of genetically engineered mice with either gain-of-function or loss-of-function mutations is the most popular technique for determining gene functions and the interrelationship between molecules in vivo. These models have provided a wealth of information about the developmental and physiological roles of oncogenes and growth factors. To date, transgenic techniques have been used extensively to study the functions of the epidermal growth factor (EGF) family. This review highlights some of the major recent findings pertinent to the EGF receptor (EGFR) and its ligands with special reference to elucidating how EGF and its related growth factors work together to regulate reproduction, growth and development. Finally, future investigations on ligand-ligand communications, EGFR and its ligands in neural stem cell research, and the mechanisms of EGFR signaling and trafficking in cells are also suggested. Received 24 May 2002; received after revision 15 July 2002; accepted 16 July 2002     

Roles of the Skp2/p27 axis in the progression of chronic nephropathy

S-phase kinase-associated protein 2 (Skp2) is an F-box protein component of the Skp/Cullin/F-box-type E3 ubiquitin ligase that targets several cell cycle regulatory proteins for degradation through the ubiquitin-dependent pathway. Skp2-mediated degradation of p27, a cyclin-dependent kinase inhibitor, is involved in cell cycle regulation. Tubular epithelial cell proliferation is a characteristic feature of renal damage that is apparent in the early stages of nephropathy. The p27 level is associated with the progression of renal injury, and increased Skp2 expression in progressive nephropathy is implicated in decreases of p27 expression. In Skp2^?/? mice, renal damage caused by unilateral ureteral obstruction (UUO) was ameliorated by p27 accumulation, mainly in tubular epithelial cells. However, the amelioration of UUO-induced renal injury in Skp2^?/? mice was prevented by p27 deficiency in Skp2^?/?/p27^?/? mice. These results suggest that the Skp2-mediated reduction in p27 is a pathogenic activity that occurs during the progression of nephropathy. Here, we discuss the roles of the Skp2/p27 axis and/or related signaling pathways/components in the progression of chronic nephropathy.     

Endocytosis mechanism of P2Y2 nucleotide receptor tagged with green fluorescent protein: clathrin and actin cytoskeleton dependence

Extracellular nucleotides exert a large number of physiological effects through activation of P2Y receptors. We expressed rat P2Y₂ (rP2Y₂) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining [Ca²⁺]_i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y₂-GFP receptor internalization. Colocalization of the internalized receptor with early endosomes, clathrin and lysosomes was detected by confocal microscopy. The inhibition of receptor endocytosis by either high-density medium or chlorpromazine in the presence of UTP indicates that the receptor was internalized by the clathrin-mediated pathway. The caveolin- mediated pathway was not involved. Targeting of the receptor from endosomes to lysosomes seems to involve the proteasome pathway, because proteasomal inhibition increased receptor recycling back to the plasma membrane.Received 8 February 2005; received after revision 18 March 2005; accepted 11 April 2005     

Peripheral erythrocyte levels, hemolysis and three vanadium compounds

Three vanadium compounds of different valence states were administered to adult mice. Two, four, and eight days following treatment of vanadium, cardiac blood was collected. The blood sample was used to ascertain the peripheral erythrocyte count (cell/mm3) and to determine the in vitro hemolytic index of erythrocytes obtained from mice treated in vivo with either the tri-, tetra-, or pentavalent vanadium compound. Data indicate that the tetravalent form was the most effective test substance in 1) promoting rupture of isolated erythrocytes compared to red cells retrieved from control mice and 2) depressing the erythrocyte count obtained from heart blood; maximum effects were manifest four days post-treatment. For all treatments there appeared to be a good correlation between the degree of vanadium-induced hemolysis and the peripheral erythrocyte count reduction following exposure to the vanadium.