Isolation of the vitellogenin-binding protein from locust ovaries

A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-β-D-glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 μg (intact ovarian membranes) to 170.9 μg (affinity purified binding protein) per mg membrane protein, which corresponds to a purification factor of 35.     

Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Specific activation of Gsalpha by an NDPK B.Gbetagamma complex in H10 cells

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.     

Prolonged blood glucose reduction in mrp-2 deficient rats (GY/TR(-)) by the glucose-6-phosphate translocase inhibitor S 3025

Chlorogenic acid derivatives are potent inhibitors of hepatic glucose production by inhibition of the glucose-6-phosphate translocase component of the hepatic glucose-6-phosphatase system. The pharmacological proof of concept was clearly demonstrated during i.v. infusion of potent derivatives (S 4048, S 3483) in rats. However, the blood glucose lowering effect of S 4048 after bolus i.v. injection lasted only 60-90 min. Plasma clearance of S 4048 was very high, and the parent compound was rapidly and efficiently excreted into the bile of Wistar and GY/TR(-) rats, indicating that mrp-2 was not involved in this hepatobiliary elimination process. About 72% of the total administered radioactivity appeared in the bile within 20 min after i.v. bolus injection of the radiolabeled analogue [(3)H]S 1743 in a Wistar rat. However, in GY/TR(-) rats the dicarboxylic analogue of S 4048, S 3025, was cleared from the plasma less rapidly than its parent compound and its biliary elimination was comparatively low. In contrast, S 3025 exhibited comparable pharmacokinetics and biliary elimination profile as S 4048 in Wistar rats, suggesting that biliary elimination of S 3025 is facilitated by mrp-2, functionally absent in GY/TR(-) rats. Targeting to mrp-2 resulted in a significantly prolonged reduction of blood glucose levels in GY/TR(-) rats after i.v. bolus administration of S 3025.     

High energy phosphate transfer by NDPK B/Gβγcomplexes - an alternative signaling pathway involved in the regulation of basal cAMP production

The activation of heterotrimeric G proteins induced by G protein coupled receptors (GPCR) is generally believed to occur by a GDP/GTP exchange at the G protein α -subunit. Nevertheless, nucleoside diphosphate kinase (NDPK) and the β-subunit of G proteins (Gβ) participate in G protein activation by phosphate transfer reactions leading to the formation of GTP from GDP. Recent work elucidated the role of these reactions. Apparently, the NDPK isoform B (NDPK B) forms a complex with β; γ; dimers in which NDPK B acts as a histidine kinase phosphorylating G#x03B2; at His266. Out of this high energetic phosphoamidate bond the phosphate can be transferred specifically onto GDP. The formed GTP binds to the G protein α -subunit and thus activates the respective G protein. Evidence is presented, that this process occurs independent of the classical GPCR-induced GTP/GTP exchange und thus contributes, e.g. to the regulation of basal cAMP synthesis in cells.     

Vitellogenin Titres in Normal and Accelerated Maturation of Gregarious-Phase Schistocerca gregaria

Isolation of vitellogenin of the Schistocerca gregaria (Forskal) in its gregarious phase was achieved by a combination of gel permeation and anion exchange chromatography. Staining for carbohydrate and lipid moieties showed that the vitellogenin is a glycolipoprotein. The vitellogenin of S. gregaria has a native molecular weight of about 700 kDa. On SDS-PAGE, the protein showed nine apoproteins of about 124, 120, 105, 60, 59, 58, 57, 53 and 34 kD. Determination of the levels of vitellogenin by ELISA in the haemolymph of maturing females showed that those exposed to mature males from 1 to 2 days after ecdysis had increased levels of vitellogenin from day 10 (81.1 ± 4.5). In contrast, females exposed to immature males or kept alone showed an increase (107.3 ± 0.9 and 70.2 ± 2.7) not until day 16 or later, respectively. These results are consistent with the accelerating effect of pheromonal emissions from mature males on the maturation of female S. gregaria.     

Body Building on Diamonds

Whereas conservative therapies aim to stall the advance of disease,regenerative medicine strives to reverse it.The capacityof most tissues to regenerate derives from stem cells,but there are a number of barriers which have to be circumvented before itwill be possible to use stem-cell-based therapies.Such therapies,however,are expected to improve human health enormously,and knowledge gained from studying stem cells in culture and in model organisms is now laying the groundwork for a new eraof regenerative medicine.One of the most prominent methods to study stem cell differentiation is to let them to form embryoidbodies.Under favourable conditions any stem cell line will form embryoid bodies.However,the mechanism of the formation ofembryoid bodies is not very well understood,and to produce them in the laboratory is in no way trivial-an important technicalbarrier in stem cell research.Recently,the embryoid body cultivation step has been successfully circumvented for the derivationof osteogenic cultures of embryonic stem cells.Here we report on a simple and reusable system to cultivate embryoid bodies inextremely short times.The method is inspired by the principles that lead to the establishment of the biomimetic triangle.     

Comparative LCA of recycled and conventional concrete for structural applications

Purpose

Construction and demolition (C&D) waste recycling has been considered to be a valuable option not only for minimising C&D waste streams to landfills but also for mitigating primary mineral resource depletion. However, the potentially higher cement demand due to the larger surface of the coarse recycled aggregates challenges the environmental benefits of recycling concrete. Furthermore, it is unclear how the environmental impacts depend on concrete mixture, cement type, aggregates composition and transport distances.

Methods

We therefore analysed the life cycle impacts of 12 recycled concrete (RC) mixtures with two different cement types and compared it with corresponding conventional concretes (CC) for three structural applications. The RC mixtures were selected according to laws, standards and construction practice in Switzerland. We compared the environmental impacts of ready-for-use concrete on the construction site, assuming equal lifetimes for recycled and conventional concrete in a full life cycle assessment. System expansion and substitution are considered to achieve the same functionality for all systems.

Results and discussion

The results show clear (～30 %) environmental benefits for all RC options at endpoint level (ecoindicator 99 and ecological scarcity). The difference is mainly due to the avoided burdens associated to reinforcing steel recycling and avoided disposal of C&D waste. Regarding global warming potential (GWP), the results are more balanced and primarily depend on the additional amount of cement needed for RC. Above 22 to 40 kg additional cement per cubic metre of concrete, RC exhibits a GWP comparable to CC. Additional transport distances above 15 km for the RC options do result in environmental impacts higher than those for CC.

Conclusions

In summary, the current market mixtures of recycled concrete in Switzerland show significant environmental benefits compared to conventional concrete and cause similar GWP, if additional cement and transport for RC are limited.

     

Through scaffolding and catalytic actions nucleoside diphosphate kinase B differentially regulates basal and β-adrenoceptor-stimulated cAMP synthesis

β-adrenoceptors (βAR) play a central role in the regulation of cAMP synthesis and cardiac contractility. Nucleoside diphosphate kinase B (NDPK B) regulates cAMP signalling by complex formation with Gβγ dimers thereby activating and stabilizing heterotrimeric G_s proteins, key transducer of βAR signals into the cell. Here, we explored the requirement of NDPK B for basal and βAR-stimulated cAMP synthesis and analysed the underlying mechanisms by comparing wild-type NDPK B (WT) and its catalytically inactive H118N mutant. Stable overexpression of both WT- and H118N-NDPK B in cardiomyocyte derived H10 cells increased the plasma membrane content of G_s and caveolin-1 and thus enhanced the isoproterenol (ISO)-stimulated cAMP-synthesis by about 2-fold. Conversely, the loss of NDPK B in embryonic fibroblasts from NDPK A/B-depleted mice was associated with a severe reduction in membranous G_s protein and carveolin-1 content causing a marked decrease in basal and ISO-induced cAMP formation. Re-expression of NDPK B, but not of NDPK A, was able to rescue this phenotype. Both, re-expression of WT- and H118N-NDPK B induced the re-appearance of G_s and caveolin-1 at the plasma membrane to a similar extent. Accordingly, WT- and H118N-NDPK B similarly enhanced ISO-induced cAMP formation. In contrast, the catalytically inactive H118N-NDPK B was less potent and less effective in rescuing basal cAMP production. Identical results were obtained in neonatal rat cardiac myocytes after siRNA-induced knockdown and adenoviral re-expression of NDPK B.Our data reveal that NDPK B regulates G_s function by two different mechanisms. The complex formation of NDPK B with G_s is required for the stabilization of the G protein content at the plasma membrane. In addition, the NDPK B-dependent phosphotransfer reaction, which requires the catalytic activity, specifically allows a receptor-independent, basal G_s activation.     

Strategy for genotoxicity testing--metabolic considerations

The report from the 2002 International Workshop on Genotoxicity Tests (IWGT) Strategy Expert Group emphasized metabolic considerations as an important area to address in developing a common strategy for genotoxicity testing. A working group convened at the 2005 4th IWGT to discuss this area further and propose practical strategy recommendations. To propose a strategy, the working group reviewed: (1) the current status and deficiencies, including examples of carcinogens "missed" in genotoxicity testing, established shortcomings of the standard in vitro induced S9 activation system and drug metabolite case examples; (2) the current status of possible remedies, including alternative S9 sources, other external metabolism systems or genetically engineered test systems; (3) any existing positions or guidance. The working group established consensus principles to guide strategy development. Thus, a human metabolite of interest should be represented in genotoxicity and carcinogenicity testing, including evaluation of alternative genotoxicity in vitro metabolic activation or test systems, and the selection of a carcinogenicity test species showing appropriate biotransformation. Appropriate action triggers need to be defined based on the extent of human exposure, considering any structural knowledge of the metabolite, and when genotoxicity is observed upon in vitro testing in the presence of metabolic activation. These triggers also need to be considered in defining the timing of human pharmaceutical ADME assessments. The working group proposed two strategies to consider; a more proactive approach, which emphasizes early metabolism predictions to drive appropriate hazard assessment; and a retroactive approach to manage safety risks of a unique or "major" metabolite once identified and quantitated from human clinical ADME studies. In both strategies, the assessment of the genotoxic potential of a metabolite could include the use of an alternative or optimized in vitro metabolic activation system, or direct testing of an isolated or synthesized metabolite. The working group also identified specific areas where more data or experiences need to be gained to reach consensus. These included defining a discrete exposure action trigger for safety assessment and when direct testing of a metabolite of interest is warranted versus the use of an alternative in vitro activation system, a universal recommendation for the timing of human ADME studies for drug candidates and the positioning of metabolite structural knowledge (through in silico systems, literature, expert analysis) in supporting metabolite safety qualification. Lastly, the working group outlined future considerations for refining the initially proposed strategies. These included the need for further evaluation of the current in vitro genotoxicity testing protocols that can potentially perturb or reduce the level of metabolic activity (potential alterations in metabolism associated with both the use of some solvents to solubilize test chemicals and testing to the guidance limit dose), and proposing broader evaluations of alternative metabolic activation sources or engineered test systems to further challenge the suitability of (or replace) the current induced liver S9 activation source.