Regulation of L-alanine dehydrogenase in Rhizobium leguminosarum bv. viciae and its role in pea nodules

Alanine dehydrogenase (AldA) is the principal enzyme with which pea bacteroids synthesize alanine de novo. In free-living culture, AMA activity is induced by carboxylic acids (succinate, malate, and pyruvate), although the best inducer is alanine. Measurement of the intracellular concentration of alanine showed that AldA contributes to net alanine synthesis in laboratory cultures. Divergently transcribed from aldA is an AsnC type regulator, aldR. Mutation of aldR prevents induction of AldA activity. Plasmid-borne gusA fusions showed that aldR is required for transcription of both aldA and aldR; hence, AldR is autoregulatory. However, plasmid fusions containing the aldA-aldR intergenic region could apparently titrate out AldR, sometimes resulting in a complete loss of AldA enzyme activity. Therefore, integrated aldR::gusA and aldA::gusA fusions, as well as Northern blotting, were used to confirm the induction of aldA activity. Both aldA and aldR were expressed in the II/III interzone and zone III of pea nodules. Overexpression of aldA in bacteroids did not alter the ability of pea plants to fix nitrogen, as measured by acetylene reduction, but caused a large reduction in the size and dry weight of plants. This suggests that overexpression of aldA impairs the ability of bacteroids to donate fixed nitrogen that the plant can productively assimilate. We propose that the role of AldA may be to balance the alanine level for optimal functioning of bacteroid metabolism rather than to synthesize alanine as the sole product of N-2 reduction.

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation

Objectives: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. Methods: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We. determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. Results: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. Conclusions: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression. (c) 2007 European Society of Cardiology. Published by Elsevier B.V. All fights reserved.

Exploring the market potential of 'local' in food systems

Local food initiatives create a niche market in many developed countries where consumer choice is being met with an expanding offering in both conventional as well as complementary retail outlets. Supermarkets in conjunction with the food service sector currently dominate food sales and consumption, and are likely to do so for the foreseeable future. However, the local food sector offers an opportunity for implementing niche marketing strategies for many businesses. Local food activities tend to be relatively independent activities and a clearer definition for “local” food would assist in consolidating this important component of the food system. Related to this, consumers would benefit from the establishment of some form of assurance system for the ‘localness’ of food. In the UK, with its well established local food market, farmers’ markets, farm shops and box schemes are currently having the largest impact in terms of total sales. Hence further research is required to confirm that support for similar business ventures in Australia would be a viable strategy for strengthening its local food systems.

The dependent variable problem in quantitative studies of Active Labour Market Programmes: uncovering hidden dynamics?

The question of what explains variation in expenditures on Active Labour Market Programs (ALMPs) has attracted significant scholarship in recent years. Significant insights have been gained with respect to the role of employers, unions and dual labour markets, openness, and partisanship. However, there remain significant disagreements with respects to key explanatory variables such the role of unions or the impact of partisanship. Qualitative studies have shown that there are both good conceptual reasons as well as historical evidence that different ALMPs are driven by different dynamics. There is little reason to believe that vastly different programs such as training and employment subsidies are driven by similar structural, interest group or indeed partisan dynamics. The question is therefore whether different ALMPs have the same correlation with different key explanatory variables identified in the literature? Using regression analysis, this paper shows that the explanatory variables identified by the literature have different relation to distinct ALMPs. This refinement adds significant analytical value and shows that disagreements are at least partly due to a dependent variable problem of ‘over-aggregation’.

Functional significance of the signal transduction pathways Akt and Erk in ovarian follicles: in vitro and in vivo studies in cattle and sheep

BACKGROUND: The intracellular signalling mechanisms that regulate ovarian follicle development are unclear; however, we have recently shown differences in the Akt and Erk signalling pathways in dominant compared to subordinate follicles. The aim of this study was to investigate the effects of inhibiting Akt and Erk phosphorylation on IGF- and gonadotropin- stimulated granulosa and theca cell function in vitro, and on follicle development in vivo. METHODS: Bovine granulosa and theca cells were cultured for six days and stimulated with FSH and/or IGF, or LH in combination with PD98059 (Erk inhibitor) and/or LY294002 (Akt inhibitor) and their effect on cell number and hormone secretion (estradiol, activin-A, inhibin-A, follistatin, progesterone and androstenedione) determined. In addition, ovarian follicles were treated in vivo with PD98059 and/or LY294002 in ewes on Day 3 of the cycle and follicles were recovered 48 hours later. RESULTS: We have shown that gonadotropin- and IGF-stimulated hormone production by granulosa and theca cells is reduced by treatment with PD98059 and LY294002 in vitro. Furthermore, treatment with PD98059 and LY294002 reduced follicle growth and oestradiol production in vivo. CONCLUSION: These results demonstrate an important functional role for the Akt and Erk signalling pathways in follicle function, growth and development.

Uncertainty and feasibility studies: an Italian case study

Uncertainty affects all aspects of the property market but one area where the impact of uncertainty is particularly significant is within feasibility analyses. Any development is impacted by differences between market conditions at the conception of the project and the market realities at the time of completion. The feasibility study needs to address the possible outcomes based on an understanding of the current market. This requires the appraiser to forecast the most likely outcome relating to the sale price of the completed development, the construction costs and the timing of both. It also requires the appraiser to understand the impact of finance on the project. All these issues are time sensitive and analysis needs to be undertaken to show the impact of time to the viability of the project. The future is uncertain and a full feasibility analysis should be able to model the upside and downside risk pertaining to a range of possible outcomes. Feasibility studies are extensively used in Italy to determine land value but they tend to be single point analysis based upon a single set of “likely” inputs. In this paper we look at the practical impact of uncertainty in variables using a simulation model (Crystal Ball ©) with an actual case study of an urban redevelopment plan for an Italian Municipality. This allows the appraiser to address the issues of uncertainty involved and thus provide the decision maker with a better understanding of the risk of development. This technique is then refined using a “two-dimensional technique” to distinguish between “uncertainty” and “variability” and thus create a more robust model.

Characterisation of the intracellular-glutamate decarboxylase system (GAD): analysis of its function, transcription and role in the acid resistance of various strains of Listeria monocytogenes

The glutamate decarboxylase (GAD) system is important for the acid resistance of Listeria monocytogenes. We previously showed that under acidic conditions, glutamate (Glt)/γ-aminobutyrate (GABA) antiport is impaired in minimal media but not in rich ones, like brain heart infusion. Here we demonstrate that this behavior is more complex and it is subject to strain and medium variation. Despite the impaired Glt/GABA antiport, cells accumulate intracellular GABA (GABA(i)) as a standard response against acid in any medium, and this occurs in all strains tested. Since these systems can occur independently of one another, we refer to them as the extracellular (GAD(e)) and intracellular (GAD(i)) systems. We show here that GAD(i) contributes to acid resistance since in a ΔgadD1D2 mutant, reduced GABA(i) accumulation coincided with a 3.2-log-unit reduction in survival at pH 3.0 compared to that of wild-type strain LO28. Among 20 different strains, the GAD(i) system was found to remove 23.11% ± 18.87% of the protons removed by the overall GAD system. Furthermore, the GAD(i) system is activated at milder pH values (4.5 to 5.0) than the GAD(e) system (pH 4.0 to 4.5), suggesting that GAD(i) is the more responsive of the two and the first line of defense against acid. Through functional genomics, we found a major role for GadD2 in the function of GAD(i), while that of GadD1 was minor. Furthermore, the transcription of the gad genes in three common reference strains (10403S, LO28, and EGD-e) during an acid challenge correlated well with their relative acid sensitivity. No transcriptional upregulation of the gadT2D2 operon, which is the most important component of the GAD system, was observed, while gadD3 transcription was the highest among all gad genes in all strains. In this study, we present a revised model for the function of the GAD system and highlight the important role of GAD(i) in the acid resistance of L. monocytogenes.