Modification of cytokinins by cauliflower microsomal enzymes

Two homozygous mutant lines of barley (Hordeum vulgare L.) R3202 (Lt1b/Lt1b) and R3004 (Lt2/Lt2), are resistant to lysine plus threonine. They contain aspartate kinase isoenzymes with lost or decreased feedback sensitivity to lysine in either isoenzyme AKII (R3202) or isoenzyme AKIII (R3004). A homozygous double mutant line (Lt1b/Lt1b, Lt2/Lt2) has now been constructed that grows vigorously on 8 millimolar lysine, 8 millimolar threonine, and 1 millimolar arginine. Both AKII and AKIII from the double mutant have altered lysine sensitivities, identical to those previously observed in R3202 and R3004, respectively. Aspartate kinase activity in extracts of leaves, roots, and the maturing endosperm of the double mutant was much less sensitive to lysine inhibition than the enzyme in comparable extracts of the parent cv Bomi, suggesting that aspartate kinase is expressed in a similar manner in different tissues of barley.

A further mutant, R2501, resistant to lysine plus threonine has now given rise to a homozygous line (Lt1a/Lt1a), which had previously not been possible. AKII isolated from the homozygous line was completely insensitive to 10 millimolar lysine; however, the combined action of 10 millimolar lysine and 0.8 millimolar S-adenosylmethionine inhibited it by 60%, demonstrating the retention of some of the regulatory characteristics of the wild type enzyme.

     

High Variation of Fluorescence Protein Maturation Times in Closely Related Escherichia coli Strains

Fluorescent proteins (FPs) are widely used in biochemistry, biology and biophysics. For quantitative analysis of gene expression FPs are often used as marking molecules. Therefore, sufficient knowledge of maturation times and their affecting factors is of high interest. Here, we investigate the maturation process of the FPs GFP and mCherry expressed by the three closely related Escherichia coli strains of the Colicin E2 system, a model system for colicinogenic interaction. One strain, the C strain produces Colicin, a toxin to which the S strain is sensitive, and against which the R strain is resistant. Under the growth conditions used in this study, the S and R strain have similar growth rates, as opposed to the C strain whose growth rate is significantly reduced due to the toxin production. In combination with theoretical modelling we studied the maturation kinetics of the two FPs in these strains and could confirm an exponential and sigmoidal maturation kinetic for GFP and mCherry, respectively. Our subsequent quantitative experimental analysis revealed a high variance in maturation times independent of the strain studied. In addition, we determined strain dependent maturation times and maturation behaviour. Firstly, FPs expressed by the S and R strain mature on similar average time-scales as opposed to FPs expressed by the C strain. Secondly, dependencies of maturation time with growth conditions are most pronounced in the GFP expressing C strain: Doubling the growth rate of this C strain results in an increased maturation time by a factor of 1.4. As maturation times can vary even between closely related strains, our data emphasize the importance of profound knowledge of individual strains'' maturation times for accurate interpretation of gene expression data.     

Immediate and Heterogeneous Response of the LiaFSR Two-Component System of Bacillus subtilis to the Peptide Antibiotic Bacitracin

Background

Two-component signal transduction systems are one means of bacteria to respond to external stimuli. The LiaFSR two-component system of Bacillus subtilis consists of a regular two-component system LiaRS comprising the core Histidine Kinase (HK) LiaS and the Response Regulator (RR) LiaR and additionally the accessory protein LiaF, which acts as a negative regulator of LiaRS-dependent signal transduction. The complete LiaFSR system was shown to respond to various peptide antibiotics interfering with cell wall biosynthesis, including bacitracin.

Methodology and Principal Findings

Here we study the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. Using quantitative fluorescence microscopy, we performed a whole population study analyzed on the single cell level. We investigated switching from the non-induced ‘OFF’ state into the bacitracin-induced ‘ON’ state by monitoring gene expression of a fluorescent reporter from the RR-regulated liaI promoter. We found that switching into the ‘ON’ state occurred within less than 20 min in a well-defined switching window, independent of the bacitracin concentration. The switching rate and the basal expression rate decreased at low bacitracin concentrations, establishing clear heterogeneity 60 min after bacitracin induction. Finally, we performed time-lapse microscopy of single cells confirming the quantitative response as obtained in the whole population analysis for high bacitracin concentrations.

Conclusion

The LiaFSR system exhibits an immediate, heterogeneous and graded response to the inducer bacitracin in the exponential growth phase.     

Risk factors of peripheral arterial disease: a case control study in Sri Lanka

Background

Peripheral artery disease (PAD) is an important global health problem and contributes to notable proportion of morbidity and mortality. This particular manifestation of systemic atherosclerosis is largely under diagnosed and undertreated. For sustainable preventive strategies in a country, it is mandatory to identify country-specific risk factors. We intended to assess the risk factors of PAD among adults aged 40–74 years.

Methods

This case control study was conducted in 2012–2013 in Sri Lanka. Seventy-nine cases and 158 controls in the age group of 40–74 years were selected for the study in order to have case to control ratio 1:2. The criterion for selecting cases and control was based on Ankle brachial pressure index (ABPI). Cases were selected from those who had ABPI 0.85 or less (ABPI ≤0.85) in either lower limb. Controls were selected from those ABPI score between 1.18 and 1.28 in both lower limbs. Only newly identified individuals with PAD were selected as cases. Controls were selected from the same geographical location and within the 5 year age group as cases.

Results

The history of diabetes mellitus more than 10 years (OR 5.8, 95% CI 2.2–14.2), history of dyslipidemia for more than 10 years (OR 4.9, 95% CI 2.1–16.2), history of hypertension for more than 10 years (OR 3.8, 95% CI 1.8–12.7) and smoking (OR 2.9, 95% CI 1.2–6.9), elevated HsCRP (OR 3.7, 95% CI 1.2–12.0) and hyperhomocysteinemia (OR 3.0, 95% CI 1.1–8.1) were revealed as country specific significant risk factor of PAD.

Conclusions

Diabetes mellitus, hypertension, dyslipidemia, smoking as well as elevated homocysteine and HsCRP found as risk factors of PAD. Longer the duration or higher level exposure to these risk factors has increased the risk of PAD. These findings emphasis the need for routine screening of PAD among patients with the identified risk factors.

     

The role of fish-poultry integration on fish growth performance,yields and economic benefits among smallholder farmers in sub-Saharan Africa,Tanzania

Aquaculture practices from sub-Saharan Africa are characterised by low production, owing to improper technology. Production can be increased through integrating fish farming with other existing on-farm activities, particularly livestock husbandry. We assessed the role of fish-poultry integration on all male Nile tilapia, Oreochromis niloticus growth performance, yields and economic benefits among smallholder farmers in sub-Saharan Africa, Tanzania. The study also compared phytoplankton species composition, abundance and biomass between the fish-poultry integration and non-integrated system. After 180 days of the experiment, all male O. niloticus cultured under fish-poultry integration exhibited significantly higher growth rates than those in the non-integrated system (p < 0.05). Gross fish yield (GFY), net fish yield (NFY) and net annual yields (NAY) obtained from fish-poultry integration were significantly higher than those from non-integrated system (p < 0.05). Partial enterprise budget analysis revealed that fish-poultry integration was more profitable than the non-integrated system. Moreover, fish-poultry integrated system produced significantly higher phytoplankton abundance and biomass than those from the non-integrated system. Results demonstrate that rural smallholder farmers can achieve higher growth rate, farm net yields and income by integrating all male O. niloticus with other on-farm activities than practising a stand-alone fish culture system.     

Bidirectional transmembrane modulation of integrin alphaIIbbeta3 conformations

Activation of blood platelets by physiological stimuli (e.g. thrombin, ADP) at sites of vascular injury induces inside-out signaling, resulting in a conformational change of the prototype integrin alphaIIbbeta3 from an inactive to an active state competent to bind soluble fibrinogen. Furthermore, ligand occupancy of alphaIIbbeta3 initiates outside-in signaling and additional conformational changes of the receptor, leading to the exposure of extracellular neoepitopes termed ligand-induced binding sites (LIBS), which are recognized by anti-LIBS monoclonal antibodies. To date, the mechanism of bidirectional transmembrane signaling of alphaIIbbeta3 has not been established. In this study, using our newly developed anti-LIBScyt1 monoclonal antibody, we showed that extracellular ligand binding to alphaIIbbeta3 on blood platelets induces a transmembrane conformational change in alphaIIbbeta3, thereby exposing the LIBScyt1 epitope in the alphaIIb cytoplasmic sequence between Lys994 and Asp1003. In addition, a point mutation at this site (P998A/P999A) renders alphaIIbbeta3 constitutively active to bind extracellular ligands, resulting in fibrinogen-dependent cell-cell aggregation. Taken collectively, these results demonstrated that the extracellular ligand-binding site and a cytoplasmic LIBS epitope in integrin alphaIIbbeta3 are conformationally and functionally coupled. Such bidirectional modulation of alphaIIbbeta3 conformation across the cell membrane may play a key role in inside-out and outside-in signaling via this integrin.     

Multiple domains within the Cauliflower mosaic virus gene VI product interact with the full-length protein

The Cauliflower mosaic virus (CaMV) gene VI product (P6) is a multifunctional protein essential for viral propagation. It is likely that at least some of these functions require P6 self-association. The work described here was performed to confirm that P6 self-associates and to identify domains involved in this interaction. Yeast two-hybrid analyses indicated that full-length P6 self-associates and that this interaction is specific. Additional analyses indicated that at least four independent domains bind to full-length P6. When a central domain (termed domain D3) was removed, these interactions were abolished. However, this deleted P6 was able to bind to the full-length wild-type protein and to isolated domain D3. Viruses lacking domain D3 were incapable of producing a systemic infection. Isolated domain D3 was capable of binding to at least two of the other domains but was unable to self-associate. This suggests that domain D3 facilitates P6 self-association by binding to the other domains but not itself. The presence of multiple domains involved in P6 self-association may help explain the ability of this protein to form the intracellular inclusions characteristic of caulimoviruses.