Prediction and prenosological diagnostics of heart diseases based on energy characteristics of acupuncture points and fuzzy logic

Many theories of reflexology use ancient concepts which do not coincide with the modern medical terminology of anatomy, physiology and biophysics. This substantially reduces the trust of physicians in reflexology methods. During this research, several mathematical models for the interaction of the internal and biological active points of meridian structures have been proposed. The analysis of these models allows the specification of a list of heart diseases for which reflex diagnostics and reflex therapy methods are most effective and also allows increasing the effectiveness of these procedures. It is shown that good results for the prediction and early diagnosis of diseases from the reaction energy of biologically active points (acupuncture points) are obtained using fuzzy logic decision making.     

FRET analysis reveals a critical conformational change within the Na,K-ATPase alpha1 subunit N-terminus during GPCR-dependent endocytosis

Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. It induces the endocytosis of plasma membrane Na,K-ATPase molecules, and this results in a reduced capacity of the cells to transport sodium. Dopamine induces the phosphorylation of Ser-18 in the alpha1-subunit of Na,K-ATPase. Fluorescence resonance energy transfer analysis of cells expressing YFP-alpha1 and beta1-CFP reveals that treatment of the cells with dopamine increases energy transfer between CFP and YFP. This is consistent with a protein conformational change that results in the N-terminal end of alpha1 moving closer to the internal face of the plasma membrane.     

Effects of the antidepressant fluoxetine on the subcellular localization of 5-HT1A receptors and SERT

Serotonin (5-HT) 5-HT(1A) autoreceptors (5-HT(1A)autoR) and the plasmalemmal 5-HT transporter (SERT) are key elements in the regulation of central 5-HT function and its responsiveness to antidepressant drugs. Previous immuno-electron microscopic studies in rats have demonstrated an internalization of 5-HT(1A)autoR upon acute administration of the selective agonist 8-OH-DPAT or the selective serotonin reuptake inhibitor antidepressant fluoxetine. Interestingly, it was subsequently shown in cats as well as in humans that this internalization is detectable by positron emission tomography (PET) imaging with the 5-HT(1A) radioligand [(18)F]MPPF. Further immunocytochemical studies also revealed that, after chronic fluoxetine treatment, the 5-HT(1A)autoR, although present in normal density on the plasma membrane of 5-HT cell bodies and dendrites, do not internalize when challenged with 8-OH-DPAT. Resensitization requires several weeks after discontinuation of the chronic fluoxetine treatment. In contrast, the SERT internalizes in both the cell bodies and axon terminals of 5-HT neurons after chronic but not acute fluoxetine treatment. Moreover, the total amount of SERT immunoreactivity is then reduced, suggesting that SERT is not only internalized, but also degraded in the course of the treatment. Ongoing and future investigations prompted by these finding are briefly outlined by way of conclusion.     

An Adenylyl Cyclase-mAKAP�� Signaling Complex Regulates cAMP Levels in Cardiac Myocytes

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein β (mAKAPβ) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPβ selectively binds type 5 AC in the heart and that mAKAPβ-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPβ-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245–340), and expression of this peptide disrupts endogenous mAKAPβ-AC association. Accordingly, disruption of mAKAPβ-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPβ complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.The formation of multimolecular protein complexes contributes to the specificity of intracellular signaling pathways, including those regulating cardiac myocyte hypertrophy. The cAMP-dependent protein kinase (PKA)³ is targeted to specific intracellular domains by protein kinase A-anchoring proteins (AKAPs) that often serve as scaffolding proteins for diverse signaling enzymes (1). In the heart, global disruption of PKA anchoring affects cardiac contractility, while the inhibited expression of individual AKAPs such as mAKAPβ or AKAP-Lbc attenuates adrenergic-induced hypertrophy of cultured neonatal myocytes (2–4). We have recently shown that specific AKAPs, namely AKAP79 and Yotiao, bind adenylyl cyclases (AC) (5, 6). However, the functional significance of AC-AKAP complexes has not been demonstrated.mAKAPβ, expressed in striated myocytes, is one of two known splice variants encoded by the single mAKAP (AKAP6) gene (7). We previously published that mAKAPβ is primarily localized to the outer membrane of the nuclear envelope via direct binding to nesprin-1α (4, 8). In cardiac myocytes, mAKAPβ serves as the scaffold for a multimolecular signaling complex that in addition to PKA includes the ryanodine receptor (RyR2), the protein phosphatases PP2A and calcineurin, phosphodiesterase 4D3 (PDE4D3), exchange protein activated by cAMP (Epac1), ERK5, and MEK5 mitogen-activated protein kinases, molecules implicated in the regulation of cardiac hypertrophy (4, 7–13). mAKAPβ complexes facilitate cross-talk between MAP kinase, calcium, and cAMP signaling pathways, permitting feedback inhibition of cAMP levels and the dynamic regulation of PKA and ERK5 activity (4, 9–13). Accordingly, mAKAPβ RNAi attenuates adrenergic and cytokine-induced hypertrophy of cultured rat neonatal ventricular myocytes (4, 11).Because mAKAPβ forms a complex with two cAMP effectors and a metabolizing enzyme for cAMP, we considered whether AC might also be an integral part of the mAKAPβ complex. We now demonstrate that type 5 adenylyl cyclase (AC5) binds directly a unique N-terminal site on mAKAPβ and is the predominant AC isoform associated with mAKAPβ in the heart. We show that AC5 bound to mAKAPβ is inhibited by PKA-dependent negative feedback. Importantly, inhibition of endogenous mAKAPβ-AC5 binding revealed the functional importance of these complexes for the regulation of cAMP-dependent myocyte hypertrophy.     

Doxorubicin cardiomyopathy-induced inflammation and apoptosis are attenuated by gene deletion of the kinin B1 receptor

Clinical use of the anthracycline doxorubicin (DOX) is limited by its cardiotoxic effects, which are attributed to the induction of apoptosis. To elucidate the possible role of the kinin B1 receptor (B1R) during the development of DOX cardiomyopathy, we studied B1R knockout mice (B1R(-/-)) by investigating cardiac inflammation and apoptosis after induction of DOX-induced cardiomyopathy. DOX control mice showed cardiac dysfunction measured by pressure-volume loops in vivo. This was associated with a reduced activation state of AKT, as well as an increased bax/bcl2 ratio in Western blots, indicating cardiac apoptosis. Furthermore, mRNA levels of the proinflammatory cytokine interleukin 6 were increased in the cardiac tissue. In DOX B1R(-/-) mice, cardiac dysfunction was improved compared to DOX control mice, which was associated with normalization of the bax/bcl-2 ratio and interleukin 6, as well as AKT activation state. These findings suggest that B1R is detrimental in DOX cardiomyopathy in that it mediates the inflammatory response and apoptosis. These insights might have useful implications for future studies utilizing B1R antagonists for treatment of human DOX cardiomyopathy.     

Pores formation on cell membranes by hederacolchiside A1 leads to a rapid release of proteins for cytosolic subproteome analysis

Hederacolchiside A1 was used to progressively permeabilize the membrane of human melanoma MEL-5 cells. Holes formation was followed by Scanning Electron Microscopy and interaction of the saponin with cholesterol and phospholipids by TOF-SIMS. 2D-LC-MS/MS and 2D-SDS-PAGE show that the release of soluble proteins into serum-free culture media increases with time. This can lead to a new rapid and efficient strategy to analyze the cytosolic subproteome and it opens the door to get information from the cytosolic compartment for clinical proteomic studies.     

Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain

The ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long-term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post-synaptic receptors. To determine the precise distribution of EphA4 and EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD-95-enriched post-synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre-synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses.     

Immunoglobulin heavy- and light-chain repertoire in splenic marginal zone lymphoma

The considerable heterogeneity in morphology, immunophenotype, genotype, and clinical behavior of splenic marginal zone lymphoma (SMZL) hinders firm conclusions on the origin and differentiation stage of the neoplastic cells. Immunoglobulin (IG) gene usage and somatic mutation patterns were studied in a series of 43 SMZL cases. Clonal IGHV-D-J rearrangements were amplified in 42/43 cases (4 cases carried double rearrangements). Among IGHV-D-J rearrangements, IGHV3 and IGHV4 subgroup genes were used with the highest frequency. Nineteen IGHV genes were unmutated (> 98% homology to the closest germline IGHV gene), whereas 27/46 were mutated. Clonal IGKV-J and IGLV-J gene rearrangements were amplified in 36/43 cases, including 31 IGKV-J (8/31 in lambda light-chain expressing cases) and 12 IGLV-J rearrangements; 9/31 IGKV and 6/12 IGLV sequences were mutated. IGKV-J and IGLV-J rearrangements used 14 IGKV and 9 IGLV different germline genes. Significant evidence for positive selection by classical T-dependent antigen was found in only 5/27 IGHV and 6/15 IGKV+IGLV mutated genes. These results provide evidence for the diverse B-cell subpopulations residing in the SMZ, which could represent physiologic equivalents of distinct SMZL subtypes. Furthermore, they indicate that in SMZL, as in other B cell malignancies, a complementarity imprint of antigen selection might be witnessed either by IGHV, IGKV, or IGLV rearranged sequences.     

Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. The magic of its chemistry and biology

1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine) is structurally 4-deamino cytidine. The increased electrophilic character of this simple aglycon endows the molecule with unique chemical and biological properties, making zebularine a versatile starting material for the synthesis of complex nucleosides and an effective inhibitor of cytidine deaminase and DNA cytosine methyltransferase. Zebularine is a stable, antitumor agent that preferentially targets cancer cells and shows activity both in vitro and in experimental animals, even after oral administration.