Metalloids in plants: A systematic discussion beyond description

Metalloids represent a wide range of elements with intermediate physiochemical properties between metals and non-metals. Many of the metalloids, like boron, selenium, and silicon are known to be essential or quasi-essential for plant growth. In contrast, metalloids viz. arsenic and germanium are toxic to plant growth. The toxicity of metalloids largely depends on their concentration within the living cells. Some elements, at low concentration, may be beneficial for plant growth and development; however, when present at high concentration, they often exert negative effects. In this regard, understanding the molecular mechanisms involved in the uptake of metalloids by roots, their subsequent transport to different tissues and inter/intra-cellular redistribution has great importance. The mechanisms of metalloids' uptake have been well studied in plants. Also, various transporters, as well as membrane channels involved in these processes, have been identified. In this review, we have discussed in detail the aspects concerning the positive/negative effects of different metalloids on plants. We have also provided a thorough account of the uptake, transport, and accumulation, along with the molecular mechanisms underlying the response of plants to these metalloids. Additionally, we have brought up the previous theories and debates about the role and effects of metalloids in plants with insightful discussions based on the current knowledge.     

β-Hydroxymyristic acid as a chemical marker to detect endotoxins in dialysis water

An analytical chemical method has been developed for determination of β-hydroxymyristic acid (β-HMA), a component of lipopolysaccharides (LPSs/endotoxins) in dialysis water. In our investigation, the β-HMA component was used as a chemical marker for endotoxin presence in dialysis water because it is available in the molecular subunit (lipid A) and responsible for toxicity. It is the most abundant saturated fatty acid in that subunit. The developed method is based on fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD–PZ). A high-performance liquid chromatographic separation of the β-HMA derivative was achieved using an octadecyl silica column in gradient elution. A wide dynamic range of β-HMA was tested and a calibration curve was constructed with accuracy of 90% and variability of less than 10%. The limits of detection and quantification obtained were 2 and 5 μM, respectively. The developed method was applied to detect endotoxins in dialysis water by alkaline hydrolysis of LPS using NaOH (0.25 M) at 60 °C for 2 h. After hydrolysis, free acid was detected as its NBD–PZ derivative using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Good recovery rates ranging from 98 to 105% were obtained for β-HMA in dialysis water.     

Single muscle fiber proteomics reveals unexpected mitochondrial specialization

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high‐sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype‐specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type‐resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.     

Synthesis of new glycyrrhetinic acid derived ring A azepanone, 29-urea and 29-hydroxamic acid derivatives as selective 11β-hydroxysteroid dehydrogenase 2 inhibitors

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2. Whereas inhibition of 11β-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11β-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. Recently, we published a series of hydroxamic acid derivatives of glycyrrhetinic acid showing high selectivity for 11β-HSD2. The most potent and selective compound is active against human 11β-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11β-HSD1. Starting from the lead compounds glycyrrhetinic acid and the hydroxamic acid derivatives, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11β-HSD1 and 11β-HSD2 in cell lysates. Here we describe novel 29-urea- and 29-hydroxamic acid derivatives of glycyrrhetinic acid as well as derivatives with the Beckman rearrangement of the 3-oxime to a seven-membered ring, and the rearrangement of the C-ring from 11-keto-12-ene to 12-keto-9(11)-ene. The combination of modifications on different positions led to compounds comprising further improved selective inhibition of 11β-HSD2 in the lower nanomolar range with up to 3600-fold selectivity.     

Pre-ejection period by radial artery tonometry supplements echo doppler findings during biventricular pacemaker optimization

Background

Biventricular (Biv) pacemaker echo optimization has been shown to improve cardiac output however is not routinely used due to its complexity. We investigated the role of a simple method involving computerized pre-ejection time (PEP) assessment by radial artery tonometry in guiding Biv pacemaker optimization.

Methods

Blinded echo and radial artery tonometry were performed simultaneously in 37 patients, age 69.1 ± 12.8 years, left ventricular (LV) ejection fraction (EF) 33 ± 10%, during Biv pacemaker optimization. Effect of optimization on echo derived velocity time integral (VTI), ejection time (ET), myocardial performance index (MPI), radial artery tonometry derived PEP and echo-radial artery tonometry derived PEP/VTI and PEP/ET indices was evaluated.

Results

Significant improvement post optimization was achieved in LV ET (286.9 ± 37.3 to 299 ± 34.6 ms, p < 0.001), LV VTI (15.9 ± 4.8 cm to 18.4 ± 5.1 cm, p < 0.001) and MPI (0.57 ± 0.2 to 0.45 ± 0.13, p < 0.001) and in PEP (246.7 ± 36.1 ms to 234.7 ± 35.5 ms, p = 0.003), PEP/ET (0.88 ± 0.21 to 0.79 ± 0.17, p < 0.001), and PEP/VTI (17.3 ± 7 to 13.78 ± 4.7, p < 0.001). The correlation between comprehensive echo Doppler and radial artery tonometry-PEP guided optimal atrioventricular delay (AVD) and optimal interventricular delay (VVD) was 0.75 (p < 0.001) and 0.69 (p < 0.001) respectively. In 29 patients with follow up assessment, New York Heart Association (NYHA) class reduced from 2.5 ± 0.8 to 2.0 ± 0.9 (p = 0.004) at 1.8 ± 1.4 months.

Conclusion

An acute shortening of PEP by radial artery tonometry occurs post Biv pacemaker optimization and correlates with improvement in hemodynamics by echo Doppler and may provide a cost-efficient approach to assist with Biv pacemaker echo optimization.     

Cell physiology rather than enzyme kinetics can determine the efficiency of cytochrome P450-catalyzed C–H-oxyfunctionalization

Cell physiology is a critical factor determining the efficiency of reactions performed by microbial biocatalysts. In order to develop an efficient biotransformation procedure for the hydroxylation of (S)-limonene to (S)-perillyl alcohol by recombinant Pseudomonas putida cells harboring the cytochrome P450 monooxygenase CYP153A6, physiological parameters were optimized. The previously reported synthesis of (S)-perillyl alcohol by P. putida GPo12 was based on complex and sensitive octane feeding strategies (van Beilen et al. in Appl Environ Microbiol 71:1737-1744, 2005), indicating the pivotal role of cell physiology. In contrast to previous findings, the screening of different carbon sources showed that glycerol and citrate are suitable alternatives to octane allowing high specific limonene hydroxylation activities. The use of P. putida KT2440 as an alternative host strain and citrate as the carbon source improved practical handling and allowed a 7.5-fold increase of the specific activity (to 22.6 U g (CDW) (-1) ). In two-liquid-phase biotransformations, 4.3 g of (S)-perillyl alcohol L (tot) (-1) were produced in 24 h, representing a sixfold improvement in productivity compared to previously reported results. It is concluded that, for selective cytochrome P450-based hydrocarbon oxyfunctionalizations by means of living microbial cells, the relationship between cell physiology and the target biotransformation is crucial, and that understanding the relationship should guide biocatalyst and bioprocess design.     

Genome-wide distribution and organization of microsatellites in plants: an insight into marker development in Brachypodium

Plant genomes are complex and contain large amounts of repetitive DNA including microsatellites that are distributed across entire genomes. Whole genome sequences of several monocot and dicot plants that are available in the public domain provide an opportunity to study the origin, distribution and evolution of microsatellites, and also facilitate the development of new molecular markers. In the present investigation, a genome-wide analysis of microsatellite distribution in monocots (Brachypodium, sorghum and rice) and dicots (Arabidopsis, Medicago and Populus) was performed. A total of 797,863 simple sequence repeats (SSRs) were identified in the whole genome sequences of six plant species. Characterization of these SSRs revealed that mono-nucleotide repeats were the most abundant repeats, and that the frequency of repeats decreased with increase in motif length both in monocots and dicots. However, the frequency of SSRs was higher in dicots than in monocots both for nuclear and chloroplast genomes. Interestingly, GC-rich repeats were the dominant repeats only in monocots, with the majority of them being present in the coding region. These coding GC-rich repeats were found to be involved in different biological processes, predominantly binding activities. In addition, a set of 22,879 SSR markers that were validated by e-PCR were developed and mapped on different chromosomes in Brachypodium for the first time, with a frequency of 101 SSR markers per Mb. Experimental validation of 55 markers showed successful amplification of 80% SSR markers in 16 Brachypodium accessions. An online database 'BraMi' (Brachypodium microsatellite markers) of these genome-wide SSR markers was developed and made available in the public domain. The observed differential patterns of SSR marker distribution would be useful for studying microsatellite evolution in a monocot-dicot system. SSR markers developed in this study would be helpful for genomic studies in Brachypodium and related grass species, especially for the map based cloning of the candidate gene(s).     

Heme oxidation in a chimeric protein of the alpha-selective Neisseriae meningitidis heme oxygenase with the distal helix of the delta-selective Pseudomonas aeruginosa

Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IXdelta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.     

Synthesis of glycyrrhetinic acid derivatives for the treatment of metabolic diseases

The effect of glycyrrhetinic acid (GA) and GA-derivatives towards 11β-hydroxysteroid dehydrogenase (11β-HSD) was investigated. Novel compounds with modifications at positions C-3, C-11 and C-29 of the GA skeleton were prepared. Single crystal X-ray diffraction data of selected substances are reported and discussed.