Residues Phe342-Asn346 of activated coagulation factor IX contribute to the interaction with low density lipoprotein receptor-related protein

When blood coagulation factor IX is converted to activated factor IX (factor IXa), it develops enzymatic activity and exposes the binding sites for both activated factor VIII and the endocytic receptor low density lipoprotein receptor-related protein (LRP). In the present study we investigated the interaction between factor IXa and LRP in more detail, using an affinity-purified soluble form of LRP (sLRP). Purified sLRP and full-length LRP displayed similar binding to factor IXa. An anti-factor IX monoclonal antibody CLB-FIX 13 inhibited factor IXa.sLRP complex formation. Both the antibody and a soluble recombinant fragment of LRP (i.e. cluster IV) interfered with factor IXa amidolytic activity, suggesting that the antibody and LRP share similar binding regions near the active site of factor IXa. Next, a panel of recombinant factor IXa variants with amino acid replacements in the surface loops bordering the active site was tested for binding to antibody CLB-FIX 13 and sLRP in a solid phase binding assay. Factor IXa variants with mutations in the region Phe(342)-Asn(346), located between the active site of factor IXa and factor VIII binding helix, showed reduced binding to both antibody CLB-FIX 13 and sLRP. Surface plasmon resonance analysis revealed that the variant with Asn(346) replaced by Asp displayed slower association to sLRP, whereas the variant with residues Phe(342)-Tyr(345) replaced by the corresponding residues of thrombin showed faster dissociation. Recombinant soluble LRP fragment cluster IV inhibited factor IXa-mediated activation of factor X with IC(50) values of 5 and 40 nm in the presence and absence of factor VIII, respectively. This inhibition thus seems to occur via two mechanisms: by interference with factor IXa.factor VIIIa complex assembly and by direct inhibition of factor IXa enzymatic activity. Accordingly, we propose that LRP may function as a regulator of blood coagulation.     

Ca2+-independent protein kinase Cs mediate heterologous desensitization of leukocyte chemokine receptors by opioid receptors

Heterologous desensitization of chemokine receptors by opioids has been considered to contribute to their immunosuppressive effects. Previous studies show that Met-enkephalin, an endogenous opioid, down-regulates chemotaxis of selected chemokine receptors via phosphorylation. In the present study, we further investigated the molecular mechanism of such cross-regulation. Our data showed that preincubation with Met-enkephalin inhibited both MIP-1 alpha-mediated chemotaxis and Ca(2+) flux of monocytes in a dose-dependent manner. The inhibitory effects were maximal using nanomolar concentrations of activating chemokines, a concentration found in physiological conditions. A decrease both in chemokine receptor affinity and in coupling efficiency between receptors and G protein were observed, which directly contributed to the desensitization effects. However, comparing with chemokines such as MIP-1 alpha and MCP-1, opioids did not elicit a calcium flux, failed to induce MIP-1 alpha receptors internalization, and mediated a less potent heterologous desensitization. We hypothesized that these differences might originate from the involvement of different protein kinase C (PKC) isotypes. In our studies, opioid-mediated down-regulation of MIP-1 alpha receptors could be blocked by the general PKC inhibitor calphostin C, but not by the calcium-dependent classic PKC inhibitor Go6976. Western blotting analysis and immunofluorescent staining further showed that only calcium-independent PKCs were activated upon opioid stimulation. Thus, opioids achieve desensitization of chemokine receptors via a unique pathway, involving only calcium-independent PKC isotypes.     

Allocation in recycling systems

‘Design for Recycling’ and dematerialization by enhancing the durability of products are major aspects of the quest for sustainable products. This article presents an LCA-based model for the integrated analyses of the product chain, its recycling systems, and its waste treatment systems at the ‘End of Life’ stage. The model is an extension of the EVR (Eco-costs/Value Ratio) model which has been published in this journal (Vogtländer et al. 2001), but can also be applied to other life cycle interpretation models, since the model as such is not restricted to the use of the eco-costs as a single indicator. The model has been developed to evaluate the design alternatives of complex products like buildings and cars. These products comprise several subsystems, each with its own special solution at the End of Life stage: Extending of the product life, object renovation, re-use of components, re-use of materials, useful application of waste materials, immobilization with and without useful applications, incineration with and without energy recovery, land fill. Since complex product systems always comprise a combination of these design alternatives, a methodology is given to calculate and allocate the eco-costs of the total system in order to select the best solution for sustainability. The methodology is characterized by:

1. A main allocation model of the recycling flow based on physical relationships,
2. a strict separation of the market value, the costs and the ecocosts in the system,
3. a main allocation model for extension of lifetime based on ‘depreciation of eco-costs’, parallel to economic depreciation.

     

B2 receptor activation reduces Erk1 and Erk2 phosphorylation induced by insulin-like growth factor-1, platelet-derived growth factor-BB,and high glucose in rat isolated glomeruli

Several experimental data document an activation of the mitogen-activated protein kinases Erk1 and Erk2 by bradykinin (BK), an agonist of the kinin B2 receptor (B2R). In contrast, other reports showed an inhibitory modulation of mitogenesis by BK. Therefore, we explored in the isolated glomeruli the effect of B2R activation on the signaling of insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-BB (PDGF-BB), and high glucose (HG), three factors that are believed to be involved in the development of glomerulosclerosis via the phosphorylation of Erk1 and Erk2. We observed that the activation of B2R negatively modulates the phosphorylation of Erk1 and Erk2 induced by IGF-1, PDGF-BB, and HG in the glomerulus. These effects are consistent with the hypothesis of a protective role for BK in the kidney during development of glomerulosclerosis and renal pathologies associated with a hyperproliferative state.     

Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.     

Discovering lactic acid bacteria by genomics

This review summarizes a collection of lactic acid bacteria that are now undergoing genomic sequencing and analysis. Summaries are presented on twenty different species, with each overview discussing the organisms fundamental and practical significance, nvironmental habitat, and its role in fermentation, bioprocessing, or probiotics. For those projects where genome sequence data were available by March 2002, summaries include a listing of key statistics and interesting genomic features. These efforts will revolutionize our molecular view of Gram–positive bacteria, as up to 15 genomes from the low GC content lactic acid bacteria are expected to be available in the public domain by the end of 2003. Our collective view of the lactic acid bacteria will be fundamentally changed as we rediscover the relationships and capabilities of these organisms through genomics.     

A novel protein-protein interaction between a G protein-coupled receptor and the phosphatase SHP-2 is involved in bradykinin-induced inhibition of cell proliferation

Mitogenic G protein-coupled receptor (GPCR) signaling has been extensively studied. In contrast, little is known about anti-mitogenic GPCR signaling. We show here that anti-mitogenic signaling of a GPCR, the bradykinin B2 receptor, involves a novel direct protein-protein interaction. The antiproliferative effect of bradykinin was accompanied by a transient increase in protein-tyrosine phosphatase activity. Using surface plasmon resonance analysis, we observed that an immunoreceptor tyrosine-based inhibitory motif (ITIM) located in the C-terminal part of the B2 receptor interacted specifically with the protein-tyrosine phosphatase SHP-2. The interaction was confirmed in primary culture renal mesangial cells by co-immunoprecipitation of a B2 receptor.SHP-2 complex. The extent of the interaction was transiently increased by stimulation with bradykinin, which was accompanied by an increase in specific SHP-2 phosphatase activity. Mutational analysis of the key ITIM residue confirmed that the B2 receptor ITIM sequence is required for interaction with SHP-2, SHP-2 activation, and the anti-mitogenic effect of bradykinin. Finally, in mesangial cells transfected with a dominant-negative form of SHP-2, bradykinin lost the ability to inhibit cell proliferation. These observations demonstrate that bradykinin inhibits cell proliferation by a novel mechanism involving a direct protein-protein interaction between a GPCR (the B2 receptor) and SHP-2.     

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.     

Structural basis for the inhibition of the biosynthesis of biotin by the antibiotic amiclenomycin

The antibiotic amiclenomycin blocks the biosynthesis of biotin by inhibiting the pyridoxal-phosphate-dependent enzyme diaminopelargonic acid synthase. Inactivation of the enzyme is stereoselective, i.e. the cis isomer of amiclenomycin is a potent inhibitor, whereas the trans isomer is much less reactive. The crystal structure of the complex of the holoenzyme and amiclenomycin at 1.8 A resolution reveals that the internal aldimine linkage between the cofactor and the side chain of the catalytic residue Lys-274 is broken. Instead, a covalent bond is formed between the 4-amino nitrogen of amiclenomycin and the C4' carbon atom of pyridoxal-phosphate. The electron density for the bound inhibitor suggests that aromatization of the cyclohexadiene ring has occurred upon formation of the covalent adduct. This process could be initiated by proton abstraction at the C4 carbon atom of the cyclohexadiene ring, possibly by the proximal side chain of Lys-274, leading to the tautomer Schiff base followed by the removal of the second allylic hydrogen. The carboxyl tail of the amiclenomycin moiety forms a salt link to the conserved residue Arg-391 in the substrate-binding site. Modeling suggests steric hindrance at the active site as the determinant of the weak inhibiting potency of the trans isomer.