Uptake of adenine by purine permeases of Coffea canephora

Purine permeases (PUPs) mediate the proton-coupled uptake of nucleotide bases and their derivatives into cytosol. PUPs facilitate uptake of adenine, cytokinins and nicotine. Caffeine, a purine alkaloid derived from xanthosine, occurs in only a few eudicot species, including coffee, cacao, and tea. Although caffeine is not an endogenous metabolite in Arabidopsis and rice, AtPUP1 and OsPUP7 were suggested to transport caffeine. In this study, we identified 15 PUPs in the genome of Coffea canephora. Direct uptake measurements in yeast demonstrated that CcPUP1 and CcPUP5 facilitate adenine – but not caffeine – transport. Adenine uptake was pH-dependent, with increased activity at pH 3 and 4, and inhibited by nigericin, a potassium–proton ionophore, suggesting that CcPUP1 and CcPUP5 function as proton-symporters. Furthermore, adenine uptake was not competitively inhibited by an excess amount of caffeine, which implies that PUPs of C. canephora have evolved to become caffeine-insensitive to promote efficient uptake of adenine into cytosol.     

Application of a metabolomic method combining one-dimensional and two-dimensional gas chromatography-time-of-flight/mass spectrometry to metabolic phenotyping of natural variants in rice

We have developed a comprehensive method combining analytical techniques of one-dimensional (1D) and two-dimensional (GC x GC) gas chromatography-time-of-flight (TOF)-mass spectrometry. This method was applied to the metabolic phenotyping of natural variants in rice for the 68 world rice core collection (WRC) and two other varieties. Ten metabolites were selected as metabolite representatives, and the selected ion current of each metabolite peak obtained from both techniques were statistically compared. Our method of combining 1D- and GC x GC-TOF/MS is useful for the metabolic phenotyping of natural variants in rice for further studies in breeding programs.     

The influence of visual display terminal use on the physical and mental conditions of administrative staff in Japan

Visual display terminals (VDT) are standard equipment for many office workers. Their use, however, may increase the risk of developing adverse conditions related to vision, the musculoskeletal system, and mental health. We carried out a survey among 3070 workers aged 18 to 67 years (mean, 39.9 years) at a prefectural administrative office, in which 76% of subjects were visual display terminal (VDT) users. We examined the relationship between duration of daily VDT use and eyestrain, neck or upper extremity pain, back pain, and mental health, and estimated the effect of breaks and rest during VDT work on these symptoms. The 12-item General Health Questionnaire (GHQ-12: total scores ranged from 0 to 12) was used to identify potential poor mental health status, and subjects with 4 or more were considered to have symptoms of psychological distress. Seventeen percent of subjects reported eyestrain, 19.1% reported upper extremity pain, 11.6% reported back pain, and 17% of subjects had GHQ-12 scores of 4 or higher. Logistic regression analysis showed that duration of daily VDT use and lack of breaks and rest during VDT work were significantly associated with eyestrain, neck or upper extremity pain, back pain, and psychological distress. In order to protect users from the adverse effects associated with VDT work, reducing daily VDT exposure, taking breaks, and rest during VDT work are important.     

The antimicrobial activity of the appetite peptide hormone ghrelin

The present study examined the antimicrobial activity of the peptide ghrelin. Both major forms of ghrelin, acylated ghrelin (AG) and desacylated ghrelin (DAG), demonstrated the same degree of bactericidal activity against Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), while bactericidal effects against Gram-positive Staphylococcus aureus (S. aureus) and Enterococcus faecalis (E. faecalis) were minimal or absent, respectively. To elucidate the bactericidal mechanism of AG and DAG against bacteria, we monitored the effect of the cationic peptides on the zeta potential of E. coli. Our results show that AG and DAG similarly quenched the negative surface charge of E. coli, suggesting that ghrelin-mediated bactericidal effects are influenced by charge-dependent binding and not by acyl modification. Like most cationic antimicrobial peptides (CAMPs), we also found that the antibacterial activity of AG was attenuated in physiological NaCl concentration (150mM). Nonetheless, these findings indicate that both AG and DAG can act as CAMPs against Gram-negative bacteria.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline dehydrogenases in hyperthermophilic archaea: distribution,function, structure,and application

Dye-linked l-proline dehydrogenase (ProDH) catalyzes the oxidation of l-proline to ∆¹-pyrroline-5-carboxylate (P5C) in the presence of artificial electron acceptors. The enzyme is known to be widely distributed in bacteria and eukarya, together with nicotinamide adenine dinucleotide (phosphate)-dependent P5C dehydrogenase, and to function in the metabolism of l-proline to l-glutamate. In addition, over the course of the last decade, three other types of ProDH with molecular compositions completely different from previously known ones have been identified in hyperthermophilic archaea. The first is a heterotetrameric αβγδ-type ProDH, which exhibits both ProDH and reduced nicotinamide adenine dinucleotide dehydrogenase activity and includes two electron transfer proteins. The second is a heterooctameric α₄β₄-type ProDH, which uses flavin adenine dinucleotide, flavin mononucleotide, adenosine triphosphate, and Fe as cofactors and creates a new electron transfer pathway. The third is a recently identified homodimeric ProDH, which exhibits the greatest thermostability among these archaeal ProDHs. This minireview focuses on the functional and structural properties of these three types of archaeal ProDH and their distribution in archaea. In addition, we will describe the specific application of hyperthermostable ProDH for use in a biosensor and for DNA sensing.     

Gene expression and characterization of a third type of dye-linked L-proline dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix

A third novel type of dye-linked L-proline dehydrogenase (LPDH) has recently been found in the hyperthermophilic archaeon, Pyrobaculum calidifontis, by Satomura et al. The gene encoding the enzyme homologue was identified in the aerobic hyperthermophilic archaeon, Aeropyrum pernix. The gene was successfully expressed in Escherichia coli, and the product was purified to homogeneity and characterized. The expressed enzyme was highly thermostable LPDH having a molecular mass of about 88 kDa and a homodimeric structure. The preferred substrate for the enzyme was L-proline with 2,6-dichloroindophenol (DCIP) as the electron acceptor. However, the enzyme did not utilize ferricyanide as the electron acceptor, in contrast to all other known LPDHs. The electrochemical determination of L-proline at concentrations from 0 to 0.7 mM was achieved by using A. pernix LPDH. A phylogenetic analysis revealed A. pernix LPDH to be clustered with the third type of LPDHs, and to be clearly separated from the clusters of previously known heterooligomeric LPDHs.