AKAP-Lbc: a molecular scaffold for the integration of cyclic AMP and Rho transduction pathways

A Kinase-anchoring proteins (AKAPs) are a family of functionally related proteins involved in the targeting of the PKA holoenzyme towards specific physiological substrates. We have recently identified a novel anchoring protein expressed in cardiomyocytes, called AKAP-Lbc, that functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) that activates the GTPase RhoA. Here, we discuss the most recent findings elucidating the molecular mechanisms and the transduction pathways involved in the regulation of the AKAP-Lbc signaling complex inside cells. We could show that AKAP-Lbc is regulated in a bi-directional manner by signals that activate or deactivate its Rho-GEF activity. Activation of AKAP-Lbc occurs in response to agonists that stimulate G proteins coupled receptors linked to the heterotrimeric G protein G12, whereas inactivation occurs through mechanisms that require phosphorylation of AKAP-Lbc by anchored PKA and subsequent recruitment of the regulatory protein 14-3-3. Interestingly, we could demonstrate that AKAP-Lbc can form homo-oligomers inside cells and that 14-3-3 can inhibit the Rho-GEF activity of AKAP-Lbc only when the anchoring protein adopts an oligomeric conformation. These findings reveal the molecular architecture of the AKAP-Lbc transduction complex and provide a mechanistic explanation of how upstream signaling pathways can be integrated within the AKAP-Lbc transduction complex to precisely modulate the activation of Rho.     

Photosystem I lacking the PSI-G subunit has a higher affinity for plastocyanin and is sensitive to photodamage

PSI-G is an 11 kDa subunit of PSI in photosynthetic eukaryotes. Arabidopsis thaliana plants devoid of PSI-G have a decreased PSI content and an increased activity of NADP⁺ photoreduction in vitro but otherwise no obvious phenotype [P.E. Jensen, L. Rosgaard, J. Knoetzel, H.V. Scheller, Photosystem I activity is increased in the absence of the PSI-G subunit. J. Biol. Chem. 277, (2002) 2798-2803.]. To investigate the biochemical basis for the increased activity, the kinetic parameters of the reaction between PSI and plastocyanin were determined. PSI-G clearly plays a role in the affinity for plastocyanin since the dissociation constant (K_D) is only 12 μM in the absence of PSI-G compared to 32 μM for the wild type. On the physiological level, plants devoid of PSI-G have a more reduced Q_A. This indicates that the decreased PSI content is due to unstable PSI rather than an adaptation to the increased activity. In agreement with this indication of decreased stability, plants devoid of PSI-G were found to be more photoinhibited both at low temperature and after high light treatment. The decreased PSI stability was confirmed in vitro by measuring PSI activity after illumination of a thylakoid suspension which clearly showed a faster decrease in PSI activity in the thylakoids lacking PSI-G. Light response of the P700 redox state in vivo showed that in the absence of PSI-G, P700 is more reduced at low light intensities. We conclude that PSI-G is involved in the binding dynamics of plastocyanin to PSI and that PSI-G is important for the stability of the PSI complex.     

Synthesis and insecticidal activity of fluorinated 2-(2,6-dichloro-4-trifluoromethylphenyl)-2,4,5,6-tetrahydrocyclopentapyrazoles

A number of fluorinated 1-aryl-tetrahydrocyclopentapyrazoles were synthesized and their insecticidal activity was evaluated. Some of the fluorinated compounds had significant insecticidal properties.     

Multiple genetic and epigenetic interacting mechanisms contribute to clonally selection of drug-resistant tumors: current views and new therapeutic prospective

Successful treatment of cancer requires a clear understanding of drug-resistance mechanism. Cancer patient are often treated with standard protocols without considering individual difference in chemosensitivity, whereas the efficacy of anticancer drug varies widely among individual patients. Since chemosensitivity involves multiple interacting factors, it is not sufficient to investigate a single gene or factor to fix chemoresistance. Along with affecting disease progression, the synergism between genetic and epigenetic abnormalities can contribute to convert a sensible tumor cell in a resistant one. Unlike genetic changes, epigenetic changes are potentially reversible. Therefore, treatment with DNA methylation inhibitors can reactivate the expression of genes improperly methylated and can reverse many aspect of cancer phenotype such as drug resistance. The demethylating agents are used in the treatment of several kind of tumor, but toxicity and the potential outcome on the normal methylation patterns have always been concern of researchers and clinicals. It is necessary to create individual chemosensitivity-chemoresistance maps in order to identify the combination of drugs for optimized treatments. An overview on genetic and epigenetic events contributing to clonally selection of chemotherapeutic-resistant tumors is discussed.     

Recombinant antibodies for the depletion of abundant proteins from human serum

The identification of biomarkers from serum or plasma is often hindered by a few proteins present at high concentrations, which may obscure less abundant proteins. Ideal serum depletion strategies would be flexible as regards the proteins to be removed, and would feature the use of reagents with long shelf-lives. In this article, we describe a novel protein depletion methodology based on the incubation of serum samples with phage-derived recombinant antibody fragments, which are able to bind to staphylococcal Protein A, and which carry a C-terminal peptide tag capable of streptavidin binding. The resulting protein-antibody complexes can be removed by simultaneous capture on Protein A and/or streptavidin resin. The depletion methodology was exemplified by the isolation of recombinant human mAb fragments specific to abundant human serum Ags and by the simultaneous depletion of albumin, immunoglobulins, alpha2-macroglobulin, hemoglobin, transferrin and haptoglobin. The depleted serum samples were analyzed by 2-DE and by gel-free MS-based methodologies, confirming the efficiency and selectivity of the depletion process. The methodology presented is modular in nature, since several recombinant antibodies can be combined in a single depletion experiment. Furthermore, antibodies do not have to be covalently coupled to a solid support facilitating long-term storage.     

Impaired intestinal and renal glucose transport in PDK-1 hypomorphic mice

The phosphoinositide-dependent kinase-1 (PDK-1) activates the serum- and glucocorticoid-inducible kinase and protein kinase B isoforms, which, in turn, are known to stimulate the renal and intestinal Na+-dependent glucose transporter 1. The present study has been performed to explore the role of PDK-1 in electrogenic glucose transport in small intestine and proximal renal tubules. To this end, mice expressing approximately 20% of PDK-1 (pdk1hm) were compared with their wild-type littermates (pdk1wt). According to Ussing chamber experiments, electrogenic glucose transport was significantly smaller in the jejunum of pdk1hm than of pdk1wt mice. Similarly, proximal tubular electrogenic glucose transport in isolated, perfused renal tubule segments was decreased in pdk1hm compared with pdk1wt mice. Intraperitoneal injection of 3 g/kg body wt glucose resulted in a similar increase of plasma glucose concentration in pdk1hm and in pdk1wt mice but led to a higher increase of urinary glucose excretion in pdk1hm mice. In conclusion, reduction of functional PDK-1 leads to impairment of electrogenic intestinal glucose absorption and renal glucose reabsorption. The experiments disclose a novel element of glucose transport regulation in kidney and small intestine.     

Isolation of highly active photosystem II core complexes with a His-tagged Cyt b559 subunit from transplastomic tobacco plants

Photosystem II (PSII) is a huge multi-protein-complex consisting, in higher plants and green algae, of the PS II core and the adjacent light harvesting proteins. In the study reported here, N-terminal His-tags were added to the plastome-encoded alpha-subunit of cytochrome b559, PsbE, in tobacco plants, thus facilitating rapid, mild purification of higher plant PSII. Biolistic chloroplast transformation was used to replace the wildtype psbE gene by His-tagged counterparts. Transgenic plants did not exhibit an obvious phenotype. However, the oxygen evolution capacity of thylakoids prepared from the mutants compared to the wildtype was reduced by 10-30% depending on the length of the His-tag, although Fv/Fm values differed only slightly. Homoplasmic F1 plants were used to isolate PSII cores complexes. The cores contained no detectable traces of LHC or PsaA/B polypeptides, but the main core subunits of PSII could be identified using immunodetection and mass spectroscopy. In addition, Psb27 and PsbS were detected. The presence of the former was presumably due to the preparation method, since PSII complexes located in the stroma are also isolated. In contrast to previous reports, PsbS was solely found as a monomer on SDS-PAGE in the PSII core complexes of tobacco.     

Anti-inflammatory effects of exercise training in the early period after myocardial infarction

The aim of this investigation was to determine the effect of exercise training on the levels of plasma cytokines and acute phase reactants in the early post acute myocardial infarction (AMI) period. Sixty patients were enrolled into this three-week cardiac rehabilitation study. The mean time from AMI was 7.08 +/- 1.60 days, and the patient mean age was 60 +/- 10 years. Subjects were randomly assigned to one of the two groups: the control group treated with standard measures, and the group with additional regular moderate-intensity exercise training. Physical activity was based on the ergospirometry test results. Apart from clinical follow-up and routine laboratory analysis we determined the levels of plasma cytokines: tumor necrosis factor (TNF-alpha), soluble TNF-alpha receptor 1 (TNF-alphaSR1), interleukin (IL)-8, IL-10, and acute phase reactants: high sensitivity C-reactive protein (hsCRP) and fibrinogen. The obtained results confirmed the hypothesis that the early post AMI period is an inflammatory state the intensity of which gradually decreases with standard treatment during the first month after AMI, while including patients into early exercise training improves their inflammatory profile by decreasing the level of acute phase reactant and TNF-alphaSR1.