Cyclic AMP formation of isolated human corpora lutea in response to HCG - interference by PGF2 alpha.

Human corpora lutea of defined ages were excised at operation, cut into pieces and incubated in the presence of HCG, PGF2 alpha and PGE2 alone or in combination. Following incubation cAMP formation in tissue and medium was determined. HCG-stimulated tissue cAMP content was most pronounced at a corpus luteum age of 7-10 days after ovulation. This stimulation was antagonized by PGF2 alpha in corpora lutea older than 6 days. PGE2 stimulated cAMP formation per se and this effect was more pronounced when HCG and PGE2 were combined. A possible role for PGF2 alpha as a luteolytic substance in the human is suggested.     

Activation of Wnt/β-Catenin Pathway in Monocytes Derived from Chronic Kidney Disease Patients

Patients with chronic kidney disease (CKD) have significantly increased morbidity and mortality resulting from infections and cardiovascular diseases. Since monocytes play an essential role in host immunity, this study was directed to explore the gene expression profile in order to identify differences in activated pathways in monocytes relevant to the pathophysiology of atherosclerosis and increased susceptibility to infections. Monocytes from CKD patients (stages 4 and 5, estimated GFR <20 ml/min/1.73 m²) and healthy donors were collected from peripheral blood. Microarray gene expression profile was performed and data were interpreted by GeneSpring software and by PANTHER tool. Western blot was done to validate the pathway members. The results demonstrated that 600 and 272 genes were differentially up- and down regulated respectively in the patient group. Pathways involved in the inflammatory response were highly expressed and the Wnt/β-catenin signaling pathway was the most significant pathway expressed in the patient group. Since this pathway has been attributed to a variety of inflammatory manifestations, the current findings may contribute to dysfunctional monocytes in CKD patients. Strategies to interfere with this pathway may improve host immunity and prevent cardiovascular complications in CKD patients.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Identification of a Potent Endothelium-Derived Angiogenic Factor

The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up₄U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up₄U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up₄U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up₄U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up₄U after stimulation with shear stress. Mean total plasma Up₄U concentrations of healthy subjects (N = 6) were sufficient to induce angiogenic and proliferative effects (1.34±0.26 nmol L^-1). In conclusion, Up₄U is a novel strong human endothelium-derived angiogenic factor.     

Cardiac muscle

Summary The ultrastructure of chicken and frog cardiac muscle are compared and then contrasted with the ultrastructure of mammalian cardiac muscle. Both chicken and frog cardiac muscle have no transverse tubules, remarkably few nexuses and no prominent M-lines. M-fibers of both animals are small (2–5 ) in diameter and contain dense granules. Chicken cardiac muscle like mammalian cardiac muscle has very well developed sarcoplasmic reticulum and couplings. The latter do not occur in frog cardiac muscle and the former is poorly developed in that muscle. Morphologic evidence is presented in the frog and chicken heart that would tend to attribute to the sarcoplasmic reticulum a transport function for electron-dense material (presumably proteinaceous) the possible significance of which is discussed. Purkinje fibers were identified in the form of a network on the endocardial surface of both atria and ventricles of chicken hearts. The topography of these fibers corresponds to that of a population of fibers in small mammalian hearts that, and unlike ventricular fibers in those animals, does not have transverse tubules.This investigation was presented, in part, at the 2nd Annual Summer Workshop of the Council on Basic Science of the American Heart Association in Mountain View, California, August 5–8, 1968; at the Gordon Conference on Myocardial Contractility in Holderness, New Hampshire, August 12–16, 1968; and at the 8th Annual Meeting of the American Society for Cell Biology in Boston, Massachusetts, November 11–13, 1968. This research was supported by grant No. 66737 from the American Heart Association, Inc. and by grant No. HE 08620 from the NIH.     

The upper cytokine-binding module and the Ig-like domain of the leukaemia inhibitory factor (LIF) receptor are sufficient for a functional LIF receptor complex.

To elucidate the function of the two cytokine-binding modules (CBM) of the leukemia inhibitory factor receptor (LIFR), receptor chimeras of LIFR and the interleukin-6 receptor (IL-6R) were constructed. Either the NH(2)-terminal (chimera RILLIFdeltaI) or the COOH-terminal LIFR CBM (chimera RILLIFdeltaII) were replaced by the structurally related CBM of the IL-6R which does not bind LIF. Chimera RILLIFdeltaI is functionally inactive, whereas RILLIFdeltaII binds LIF and mediates signalling as efficiently as the wild-type LIFR. Deletion mutants of the LIFR revealed that both the NH(2)-terminal CBM and the Ig-like domain of the LIFR are involved in LIF binding, presumably via the LIF site III epitope. The main function of the COOH-terminal CBM of the LIFR is to position the NH(2)-terminal CBM and the Ig-like domain, so that these can bind to LIF. In analogy to a recently published model of the IL-6R complex, a model of the active LIFR complex is suggested which positions the COOH-terminal CBM at LIF site I and the NH(2)-terminal CBM and the Ig-like domain at site III. An additional contact is postulated between the Ig-like domain of gp130 and the NH(2)-terminal CBM of the LIFR.     

TF — A novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP

BackgroundAbnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease.MethodsWe screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC₅₀ and K_i values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot.ResultsThe best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC₅₀ (29 nM) and K_i (15 nM) values. TF inhibited only seven out of 61 human kinases tested (> 70% inhibition). Incubation of LNCaP cells with 50 μM TF for 48 h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis.General significanceIn many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.