Seasonal variations in the responses of glycolytic intermediates of human erythrocytes to acute cold exposure

Seven male students were studied to observe the effects of acute cold exposure (at 10°C for 60 min) on erythrocyte concentrations of glycolytic intermediates in summer and in winter. The subjects shivered slightly but frankly in both experiments. Significant decreases were observed in the concentrations of pyruvate and lactate during body cooling in summer, but not in winter. The lactate concentration remained significantly reduced 15 min after cold exposure. After 60 min of cold exposure in summer, a negative crossover point appeared to exist between phosphoenolpyruvate and pyruvate and erythrocyte pyruvate kinase activity showed a significant decrease. No seasonal difference was observed in the initial control values of the intermediates measured. From these results and the fact that glucose, pyruvate and lactate are evenly distributed between erythrocytes and plasma, it is likely that erythrocytes and skeletal muscles need less fuel substrate, glucose during cold exposure in winter than in summer, suggesting that an increased economy of energy for homeostasis is achieved.     

Effect of food restriction on cold adaptability of rats

To determine the role of the nutritional state in nonshivering thermogenesis during cold adaptation, cold adaptability was compared between cold-adapted (5 degrees C for 4-5 weeks) rats fed ad libitum and cold-adapted rats pair fed with warm controls having the same food intake. Cold-adapted pair-fed rats suffered a significant loss in body weight during cold exposure. However, brown adipose tissue (BAT) in both cold-adapted ad libitum fed and cold-adapted pair-fed rats was enlarged to the same extent as compared with that in control rats. Fat-free dry matter in BAT also increased in cold-adapted ad libitum fed and cold-adapted pair-fed rats to the same extent. Cold tolerance as assessed by the change in the colonic temperature at -5 degrees C was improved relative to control rats and was the same for cold-adapted ad libitum fed and cold-adapted pair-fed rats. Nonshivering thermogenesis as estimated by the noradrenaline-induced increase in oxygen consumption was significantly greater in the cold-exposed rats and there was no significant difference between cold-adapted ad libitum fed and cold-adapted pair-fed rats. These results suggest that an improved cold tolerance by means of nonshivering thermogenesis in brown adipose tissue is closely related to the low temperature itself but not the increased food intake which occurred in the cold.     

Interleukin-4 as a potent inhibitor of bone resorption

A possible role of interleukin-4 (IL-4) in the regulation of bone turnover was assessed by employing a 45Ca prelabeled-fetal mouse long bone culture system. IL-4 inhibited the bone resorption stimulated by parathyroid hormone (PTH), PTH related protein (PTHrP), 1 alpha, 25, dihydroxy-vitamin D3 [1 alpha, 25 (OH)2 D3], interleukin-1 alpha and - 1 beta (IL-1 alpha, IL-1 beta) and prostaglandin E2 (PGE2). Anti-IL-4 on monoclonal antibody abolished the inhibitory effect of IL-4 on the bone resorption. These results suggest that IL-4 may play an important role on the inhibitory regulation of bone resorption.     

Enzymatic Formation of β-Citraurin from β-Cryptoxanthin and Zeaxanthin by Carotenoid Cleavage Dioxygenase4 in the Flavedo of Citrus Fruit

In this study, the pathway of β-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of β-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of β-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of β-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved β-cryptoxanthin and zeaxanthin at the 7,8 or 7′,8′ position. But other carotenoids tested in this study (lycopene, α-carotene, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of β-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of β-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.Carotenoids, a diverse group of pigments widely distributed in nature, fulfill a variety of important functions in plants and play a critical role in human nutrition and health (Schwartz et al., 1997; Cunningham and Gantt, 1998; Havaux, 1998; Krinsky et al., 2003; Ledford and Niyogi, 2005). The pathway of carotenoid biosynthesis has been well documented in various plant species, including Arabidopsis (Arabidopsis thaliana; Park et al., 2002), tomato (Lycopersicon esculentum; Isaacson et al., 2002), pepper (Capsicum annuum; Bouvier et al., 1998), citrus (Citrus spp.; Kato et al., 2004, 2006; Rodrigo et al., 2004; Rodrigo and Zacarías, 2007; Kato, 2012; Zhang et al., 2012a), and apricot (Prunus armenaica; Kita et al., 2007). Genes encoding the enzymes in the carotenoid biosynthetic pathway have been cloned, and their expression profiles have also been characterized (Fig. 1). As carotenoids contain a series of conjugated double bonds in the central chain, they can be oxidatively cleaved in a site-specific manner (Mein et al., 2011). The oxidative cleavage of carotenoids not only regulates their accumulation but also produces a range of apocarotenoids (Walter et al., 2010). In higher plants, many different apocarotenoids derive from the cleavage of carotenoids and have important metabolic functions, such as plant hormones, pigments, aroma and scent compounds, as well as signaling compounds (Fig. 1). A well-known example is abscisic acid, which is a C₁₅ compound derived from the cleavage of the 11,12 double bond of 9-cis-violaxanthin and 9′-cis-neoxanthin (Schwartz et al., 1997; Tan et al., 1997; Cutler and Krochko, 1999; Chernys and Zeevaart, 2000; Giuliano et al., 2003).Open in a separate windowFigure 1.Carotenoid and apocarotenoid metabolic pathway in plants. GGPP, Geranylgeranyl diphosphate. Enzymes, listed here from top to bottom, are named according to the designation of their genes: PSY, phytoene synthase; PDS, Phytoene desaturase; ZDS, ζ-carotene desaturase; ZISO, 15-cis-ζ-carotene isomerase; CRTISO, carotenoid isomerase; LCYb, lycopene β-cyclase; LCYe, lycopene ε-cyclase; HYe, ε-ring hydroxylase; HYb, β-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin deepoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase.Carotenoid cleavage dioxygenases (CCDs) are a group of enzymes that catalyze the oxidative cleavage of carotenoids (Ryle and Hausinger, 2002). CCDs are nonheme iron enzymes present in plants, bacteria, and animals. In plants, CCDs belong to an ancient and highly heterogenous family (CCD1, CCD4, CCD7, CCD8, and 9-cis-epoxycarotenoid dioxygenases [NCEDs]). The similarity among the different members is very low apart from four strictly conserved His residues and a few Glu residues (Kloer and Schulz, 2006; Walter et al., 2010). In Arabidopsis, the CCD family contains nine members (CCD1, NCED2, NCED3, CCD4, NCED5, NCED6, CCD7, CCD8, and NCED9), and orthologs in other plant species are typically named according to their homology with an Arabidopsis CCD (Huang et al., 2009). In our previous study, the functions of CitCCD1, CitNCED2, and CitNCED3 were investigated in citrus fruits (Kato et al., 2006). The recombinant CitCCD1 protein cleaved β-cryptoxanthin, zeaxanthin, and all-trans-violaxanthin at the 9,10 and 9′,10′ positions and 9-cis-violaxanthin at the 9′,10′ position. The recombinant CitNCED2 and CitNCED3 proteins cleaved 9-cis-violaxanthin at the 11,12 position to form xanthoxin, a precursor of abscisic acid (Kato et al., 2006). To date, information on the functions of other CCDs in citrus fruits remains limited, while the functions of CCD7 and CCD8, as well as NCED5, NCED6, and NCED9, in Arabidopsis have been characterized (Kloer and Schulz, 2006; Walter et al., 2010). In Arabidopsis, CCD7 cleaves all-trans-β-carotene at the 9′,10′ position to form all-trans-β-apo-10′-carotenal. All-trans-β-apo-10′-carotenal is further shortened by AtCCD8 at the 13,14 position to produce β-apo-13-carotenone (Alder et al., 2012). NCED5, NCED6, and NCED9 cleave 9-cis-violaxanthin at the 11,12 position to form xanthoxin (Tan et al., 2003). Compared with other CCDs, the function of CCD4 is poorly understood. In Chrysanthemum morifolium, CmCCD4a contributed to the white color formation by cleaving carotenoids into colorless compounds (Ohmiya et al., 2006). Recently, it has been reported that CsCCD4, CmCCD4a, and MdCCD4 could cleave β-carotene to yield β-ionone (Rubio et al., 2008; Huang et al., 2009).β-Citraurin, a C₃₀ apocarotenoid, is a color-imparting pigment responsible for the reddish color of citrus fruits (Farin et al., 1983). In 1936, it was first discovered in Sicilian oranges (Cual, 1965). In citrus fruits, the accumulation of β-citraurin is not a common event; it is only observed in the flavedos of some varieties during fruit ripening. The citrus varieties accumulating β-citraurin are considered more attractive because of their red-orange color (Ríos et al., 2010). Although more than 70 years have passed since β-citraurin was first identified, the pathway of its biosynthesis is still unknown. As its structure is similar to that of β-cryptoxanthin and zeaxanthin, β-citraurin was presumed to be a degradation product of β-cryptoxanthin or zeaxanthin (Oberholster et al., 2001; Rodrigo et al., 2004; Ríos et al., 2010; Fig. 1). To date, however, the specific cleavage reaction producing β-citraurin has not been elucidated. In this study, we found that the CitCCD4 gene was involved in the synthesis of β-citraurin, using two citrus varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. To confirm the role of the CitCCD4 gene further, functional analyses of the CitCCD4 enzyme were performed in vivo and in vitro. Additionally, the regulation of β-citraurin content and CitCCD4 gene expression in response to ethylene and red light-emitting diode (LED) light treatments was also examined. This study, to our knowledge, is the first to investigate the biosynthesis of β-citraurin in citrus fruits. The results might provide new strategies to enhance the nutritional and commercial qualities of citrus fruits.     

Chemical Synthesis and Characterization of α-N-Octanoyl and Other α-N-Acyl Colistin Nonapeptide Derivatives

Eight α-N-acyl colistin nonapeptide derivatives including three aliphatic, four aromatic and one alicyclic derivatives were synthesized by the reaction of colistin nonapeptide with corresponding acid chlorides. This acylation reaction was carried out under the condition kept restrictedly at pH 5,0 in order to introduce an acyl group only to α-amino group but not to γ-amino group existing in a colistin nonapeptide molecule. Synthetic method and several physico-chemical natures of these acyl colistin nonapeptide derivatives are given in this paper.

All of the acylated derivatives thus synthesized exhibited characteristic antimicrobial activities. Antimicrobial spectra were substantially based upon a gram-negative type and not so much altered by chemical structures of acyl groups which were considerably differentiated from each other such as cyclic or chain form. Thus, more possible response of carbon size than its structure to the antimicrobial effectiveness was inferred. In spite of almost no toxicity and feeble antimicrobial activity of colistin nonapeptide itself, these acylated colistin nonapeptide derivatives showed a toxicity against mice at a dose of 16.9~70 mg/kg in LD₅₀, which, however, was inferior to the toxicity of colistin sulfate, possibly correspondent to their much weaker antimicrobial activities, as a whole. Hence, it seems likely that acyl part of these acylated colistin nonapeptide derivatives including that of colistin is seriously responsible for the biological activities.     

Similarity of tetracycline resistance genes isolated from fish farm bacteria to those from clinical isolates

Tetracycline-resistant (Tet(r)) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet(r) gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet(r) genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet(r) strains transferred Tet(r) genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet(r) strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.     

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: conditional gene expression at near physiological levels

Using Multisite Gateway five-DNA-fragment constructs vectors that enable expression of two tandemly situated cDNAs on a single plasmid were developed. Heterologous protein production in cells was achieved by modulating respective cDNA expression to pre-determined and different levels. Optimization of cDNA expression at near physiological protein levels was achieved using promoters from four cell cycle-dependent genes. In comparison with conventionally available promoters, EF-1alpha or CMV, the promoters used in this study were able to modulate cDNA expression levels over a magnitude of approximately 10 or 100-fold, respectively. In transiently transfected cells, two different proteins (CPalpha1 and CPbeta2), which form a heterodimer, each labeled with a different-colored fluorescent protein, were successfully synthesized at pre-determined levels from their respective cDNAs. The above vectors were designed to contain an FRT/Flp recombination site for integration onto chromosomes and for establishment of stable clones in HeLa cells by site-specific recombination. In the stable transformant cells produced only about 4% of the protein production levels measured in the transiently transformed cells. The biological significance of these observations is discussed.