Analysis for Antigenic Similarities Among the 30S Ribosomal Proteins of Escherichia coli

Antiserum was made against a single 30S protein, 30S-7. The amount of complement fixed by total 30S protein in the presence of this antiserum indicated that protein 30S-7 was the major antigen in the mixture of proteins. Each of the 30S ribosomal proteins was tested for cross-reactivity with anti-30S-7. This was done by determining if any of the other 30S proteins inhibited complement fixation by 30S-7. None of the other 30S proteins was found to inhibit complement fixation by 30S-7, indicating that 30S-7 is antigenically distinct from the other proteins.     

Unique Protein Moieties for 30S and 50S Ribosomes of Escherichia coli

Each major component of the proteins of 30S ribosomes from Escherichia coli was compared with the proteins of 50S ribosomes. The comparisons were done by using polyacrylamide gel electrophoresis in urea with differentially labeled proteins. The data show that no major protein is common to both ribosomes.     

Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine,Pyrimidine, and Cytokinin Ribosides

We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.Nucleoside hydrolases or nucleoside N-ribohydrolases (NRHs; EC 3.2.2.-) are glycosidases that catalyze the cleavage of the N-glycosidic bond in nucleosides to enable the recycling of the nucleobases and Rib (Fig. 1A). The process by which nucleosides and nucleobases are recycled is also known as salvaging and is a way of conserving energy, which would otherwise be needed for the de novo synthesis of purine- and pyrimidine-containing compounds. During the salvage, bases and nucleosides can be converted into nucleoside monophosphates by the action of phosphoribosyltransferases and nucleoside kinases, respectively, and further phosphorylated into nucleoside diphosphates and triphosphates (Moffatt et al., 2002; Zrenner et al., 2006; Fig. 1B). Uridine kinase and uracil phosphoribosyl transferase are key enzymes in the pyrimidine-salvaging pathway in plants (Mainguet et al., 2009; Chen and Thelen, 2011). Adenine phosphoribosyltransferase and adenosine kinase (ADK) are important in purine salvaging (Moffatt and Somerville, 1988; Moffatt et al., 2002), and their mutants cause reductions in fertility or sterility, changes in transmethylation, and the formation of abnormal cell walls. In addition, both enzymes were also reported to play roles in cytokinin metabolism (Moffatt et al., 1991, 2000; von Schwartzenberg et al., 1998; Schoor et al., 2011). Cytokinins (N⁶-substituted adenine derivatives) are plant hormones that regulate cell division and numerous developmental events (Mok and Mok, 2001; Sakakibara, 2006). Cytokinin ribosides are considered to be transport forms and have little or no activity.Open in a separate windowFigure 1.A, Scheme of the reactions catalyzed by plant NRHs when using purine (inosine), pyrimidine (uridine), and cytokinin (iPR) ribosides as the substrates. B, Simplified schematic overview of cytokinin, purine, and pyrimidine metabolism in plants. The diagram is adapted from the work of Stasolla et al. (2003) and Zrenner et al. (2006) with modifications. The metabolic components shown are as follows: 1, cytokinin nucleotide phosphoribohydrolase; 2, adenine phosphoribosyltransferase; 3, adenosine kinase; 4, 5′-nucleotidase; 5, adenosine phosphorylase; 6, purine/pyrimidine nucleoside ribohydrolase; 7, cytokinin oxidase/dehydrogenase; 8, AMP deaminase; 9, hypoxanthine phosphoribosyltransferase; 10, inosine kinase; 11, inosine-guanosine phosphorylase; 12, IMP dehydrogenase; 13, xanthine dehydrogenase; 14, 5′-nucleotidase; 15, GMP synthase; 16, hypoxanthine-guanine phosphoribosyltransferase; 17, guanosine deaminase; 18, guanine deaminase; 19, guanosine kinase; 20, uracil phosphoribosyltransferase; 21, uridine cytidine kinase; 22, pyrimidine 5′-nucleotidase; 23, cytidine deaminase; 24, adenosine/adenine deaminase. CK, Cytokinin; CKR, cytokinin riboside; CKRMP, cytokinin riboside monophosphate.NRHs are metalloproteins first identified and characterized in parasitic protozoa such as Trypanosoma, Crithidia, and Leishmania species that rely on the import and salvage of nucleotide derivatives. They have since been characterized in other organisms such as bacteria, yeast, and insects (Versées and Steyaert, 2003) but never in mammals (Parkin et al., 1991). They have been divided into four classes based on their substrate specificity: nonspecific NRHs, which hydrolyze inosine and uridine (IU-NRHs; Parkin et al., 1991; Shi et al., 1999); purine-specific inosine/adenosine/guanosine NRHs (Parkin, 1996); the 6-oxopurine-specific guanosine/inosine NRHs (Estupiñán and Schramm, 1994); and the pyrimidine nucleoside-specific cytidine/uridine NRHs (CU-NRHs; Giabbai and Degano, 2004). All NRHs exhibit a stringent specificity for the Rib moiety and differ in their preferences regarding the nature of the nucleobase. Crystal structures are available for empty NRH or in complex with inhibitors from Crithidia fasciculata (CfNRH; Degano et al., 1998), Leishmania major (LmNRH; Shi et al., 1999), and Trypanosoma vivax (TvNRH; Versées et al., 2001, 2002). The structures of two CU-NRHs from Escherichia coli, namely YeiK (Iovane et al., 2008) and YbeK (rihA; Muzzolini et al., 2006; Garau et al., 2010), are also available. NRHs are believed to catalyze N-glycosidic bond cleavage by a direct displacement mechanism. An Asp from a conserved motif acts as a general base and abstracts a proton from a catalytic water molecule, which then attacks the C1′ atom of the Rib moiety of the nucleoside. Kinetic isotope-effect studies on CfNRH (Horenstein et al., 1991) showed that the substrate’s hydrolysis proceeds via an oxocarbenium ion-like transition state and is preceded by protonation at the N7 atom of the purine ring, which lowers the electron density on the purine ring and destabilizes the N-glycosidic bond. A conserved active-site His is a likely candidate for this role in IU-NRHs and CU-NRHs. In the transition state, the C1′-N9 glycosidic bond is almost 2 Å long, with the C1′ atom being sp² hybridized while the C3′ atom adopts an exo-conformation, and the whole ribosyl moiety carries a substantial positive charge (Horenstein et al., 1991).Several NRH enzymes have been identified in plants, including a uridine-specific NRH from mung bean (Phaseolus radiatus; Achar and Vaidyanathan, 1967), an inosine-specific NRH (EC 3.2.2.2) and a guanosine-inosine-specific NRH, both from yellow lupine (Lupinus luteus; Guranowski, 1982; Szuwart et al., 2006), and an adenosine-specific NRH (EC 3.2.2.7) from coffee (Coffea arabica), barley (Hordeum vulgare), and wheat (Triticum aestivum; Guranowski and Schneider, 1977; Chen and Kristopeit, 1981; Campos et al., 2005). However, their amino acid sequences have not been reported so far. A detailed study of the NRH gene family from Arabidopsis (Arabidopsis thaliana) has recently been reported (Jung et al., 2009, 2011). The AtNRH1 enzyme exhibits highest hydrolase activity toward uridine and xanthosine. It can also hydrolyze the cytokinin riboside N⁶-(2-isopentenyl)adenosine (iPR), which suggests that it may also play a role in cytokinin homeostasis. However, Riegler et al. (2011) analyzed the phenotypes of homozygous nrh1 and nrh2 single mutants along with the homozygous double mutants and concluded that AtNRHs are probably unimportant in cytokinin metabolism.Here, we identify and characterize plant IU-NRHs from two different model organisms, Physcomitrella patens and maize (Zea mays), combining structural, enzymatic, and in planta functional approaches. The moss P. patens was chosen to represent the bryophytes, which can be regarded as being evolutionarily basal terrestrial plants, and is suitable for use in developmental and metabolic studies (Cove et al., 2006; von Schwartzenberg, 2009), while maize is an important model system for cereal crops. We report the crystal structures of NRH enzymes from the two plant species, PpNRH1 and ZmNRH3. Based on these structures, we performed site-directed mutagenesis experiments and kinetic analyses of point mutants of PpNRH1 in order to identify key residues involved in nucleobase interactions and catalysis. To analyze the physiological role of the PpNRHs, single knockout mutants were generated. NRH deficiency caused significant changes in the levels of purine, pyrimidine, and cytokinin metabolites relative to those seen in the wild type, illustrating the importance of these enzymes in nucleoside and cytokinin metabolism.     

Binge Drinking and Blood Pressure: Cross-Sectional Results of the HAPIEE Study

Objectives

To investigate whether binge drinking pattern influences blood pressure independently from drinking volume or whether it modifies the effect of volume of drinking.

Methods

We used cross-sectional data from population samples of 7559 men and 7471 women aged 45–69 years in 2002-05, not on antihypertensive medication, from Russia, Poland and Czech Republic. Annual alcohol intake, drinking frequency and binge drinking (≥100 g in men and ≥60 g in women in one session at least once a month) were estimated from graduated frequency questionnaire. Blood pressure was analysed as continuous variables (systolic and diastolic pressure) and a binary outcome (≥140/90 mm Hg).

Results

In men, annual alcohol intake and drinking frequency were strongly associated with blood pressure. The odds ratio of high blood pressure for binge drinking in men was 1.62 (95% CI 1.45–1.82) after controlling for age, country, body mass index, education and smoking; additional adjustment for annual alcohol intake reduced it to 1.20 (1.03–1.39). In women, the fully adjusted odds ratio of high blood pressure for binge drinking was 1.31 (1.05–1.63). Binge drinking did not modify the effect of annual alcohol intake. Consuming alcohol as wine, beer or spirits had similar effects.

Conclusions

The results suggest that the independent long-term effect of binge drinking was modest, that binge drinking did not modify the effect of alcohol intake, and that different alcoholic beverages had similar effects on blood pressure.     

Anti-cancer activities of cytokinin ribosides

Phytochemistry Reviews - Cytokinins are plant hormones and play essential roles in regulating plant growth and development. They also have diverse pharmacological effects in animals and humans....     

How Do Different Watering Regimes Affect the Growth,Chlorophyll Fluorescence,Phytohormone, and Phenolic Acid Content of Greenhouse-Grown Ceratotheca triloba?

We evaluated the effect of different watering regimes on the growth, chlorophyll fluorescence, phytohormones, and phenolic acids in Ceratotheca triloba (Bernh.) Hook.f., a commonly consumed African indigenous leafy vegetable. The study was conducted in the greenhouse under different watering regimes [seven (daily); three (thrice); two (twice); one (once) day(s) per week] for a period of 2 and 4-months. In each pot (7.5 cm diameter; 150 ml volume), 50 ml of water was applied per treatment. At the end of the experiment, plant growth, chlorophyll fluorescence, phytohormones, and phenolic acids were determined. A decrease in water availability resulted in a consistent decline in plant growth after a 4-month growth period. The severity of reduced water availability was more noticeable in plants watered once a week with a 1.4-fold reduction in growth and quantum efficiency of PSII (Fv/Fm) value of 0.80. The significant decline in growth and chlorophyll fluorescence was probably due to the increased production of abscisic acid (ABA) and cytokinin (CK) content together with the detected phytohormones in plants with restricted water supply. Furthermore, plants watered once a week had a trade-off between growth and phenolic acid production, with significantly higher (threefolds) concentrations of vanillic, ferulic, caffeic, and 4-coumaric acids in 4-month-old plants. Even though C. triloba grew best in well-watered soil, the plant had the potential to adapt and survive in soils with limited water supply for longer periods of growth. These findings suggest that regulation of phytohormones and phenolic acids played an important role in improving the growth of C. triloba under limited water conditions.

     

Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.     

Identification of new aromatic cytokinins in Arabidopsis thaliana and Populus x canadensis leaves by LC-(+)ESI-MS and capillary liquid chromatography/frit-fast atom bombardment mass spectrometry

A search for naturally occurring aromatic cytokinins (ARCKs) in Arabidopsis thaliana plants and Populus x canadensis leaves led to the discovery of four new plant hormone substances: 6-(2-methoxybenzylamino)purine (ortho-methoxytopolin, MeoT), 6-(3-methoxybenzylamino)purine (meta-methoxytopolin, MemT) (Fig. 1) and their 9-beta-D-ribofuranosyl derivatives. These substances were identified by liquid chromatography electrospray ionization mass spectrometry [LC (+)ESI-MS] and capillary-liquid chromatography/frit-fast atom bombardment-mass spectrometry [CapLC/frit-FAB-MS] after pre-column derivatization. The chemical structures were subsequently confirmed by chemical synthesis. Because of lack of heavy labelled internal standards, the endogenous levels of methoxytopolins in A. thaliana plants, Populus x canadensis leaves and samples derived from cultures of Agrobacterium tumefaciens strain GV3101 were determined by enzyme-linked immunosorbent assay (ELISA) of HPLC-fractionated extracts. While the levels of MeoT, MemT and their ribosides in A. thaliana shoots and Populus x canadensis leaves were relatively low (approximately 0.25-10 pmol g-1 FW for MeoT and MemT, respectively), the A. tumefaciens strain produced up to 600 times more of the newly identified substances. Cytokinin activity of methoxytopolines was demonstrated in three bioassays testing their ability to stimulate tobacco callus growth, to delay chlorophyll degradation in excised wheat leaves, and to induce betacyanin synthesis in Amaranthus caudatus var. atropurpurea cotyledons. Notably, their anti-senescing activity in the wheat leaf assay exceeded that of BAP and Z by almost 200%. Methoxytopolins are proposed to be new members of the biologically active aromatic cytokinin family, which might have specific physiological functions.