Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.     

Association between Plasma Neutrophil Gelatinase Associated Lipocalin Level and Obstructive Sleep Apnea or Nocturnal Intermittent Hypoxia

Background

Both obstructive sleep apnea (OSA) and a novel lipocalin, neutrophil gelatinase associated lipocalin (Ngal), have been reported to be closely linked with cardiovascular disease and loss of kidney function through chronic inflammation. However, the relationship between OSA and Ngal has never been investigated.

Objectives

To evaluate the relationship between Ngal and OSA in clinical practice.

Methods

In 102 patients, polysomnography was performed to diagnose OSA and plasma Ngal levels were measured. The correlations between Ngal levels and OSA severity and other clinical variables were evaluated. Of the 46 patients who began treatment with continuous positive airway pressure (CPAP), Ngal levels were reevaluated after three months of treatment in 25 patients.

Results

The Ngal level correlated significantly with OSA severity as determined by the apnea hypopnea index (r = 0.24, p = 0.01) and 4% oxygen desaturation index (ODI) (r = 0.26, p = 0.01). Multiple regression analysis showed that the Ngal level was associated with 4%ODI independently of other clinical variables. Compliance was good in 13 of the 25 patients who used CPAP. Although the OSA (4%ODI: 33.1±16.7 to 1.1±1.9/h, p<0.01) had significantly improved in those with good compliance, the Ngal levels were not significantly changed (60.5±18.1 before CPAP vs 64.2±13.9 ng/ml after CPAP, p = 0.27).

Conclusions

Plasma Ngal levels were positively associated with the severity of OSA. However, the contribution rate of OSA to systemic Ngal secretion was small and changes in Ngal levels appeared to be influenced largely by other confounding factors. Therefore, it does not seem reasonable to use the Ngal level as a specific biomarker of OSA in clinical practice.     

Rheological Properties of Succinoglucan 10C3 from Alcaligenes faecalis var. myxogenes

The H-, Na- and Ca-forms of succinoglucan 10C3 form homogenous dispersions in water. The viscosities of 1% aqueous dispersion of the H- and Ca-forms were 1560 and 2500 centipoises respectively at 30°C. The viscosity of the Na-form, which is extremely low, was increased greatly by an addition of many inorganic salts and especially the Fe³⁺ and Al³⁺ iones. The viscosity of the Ca-form did not change by the presence of sodium chloride or calcium chloride.

The viscosities of Ca-form, and Na-form in the presence of 1% sodium chloride became near zero at 65.5 °C and 69.5°C, respectively. When the dispersions were kept at 70°C or 90°C for ten minutes, the viscosities of the H-form, the Na-form, and the Ca-form in the presence of sodium chloride increased greatly. The viscosity of the Ca-form and that of the Na-form in the presence of 1% sodium chloride were essentially independent of pH.     

The dissociation between the plasma levels of aminopeptidases A and B in spontaneously hypertensive rats

We performed a longitudinal study for 20 weeks on spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKR) to determine the relationship between peptide metabolism and the age-dependent increase in blood pressure. In both SHR and WKR, the plasma level of aminopeptidase A (AP-A) clearly showed an age-dependent decrease. The plasma level of aminopeptidase B paralleled that of AP-A in WKR, but such an age-dependency was not observed in SHR, thus showing a dissociation between the two aminopeptidases. With age in both strains, the level of angiotensin-converting enzyme tended to decrease, while that of kallikrein activity tended to increase. In addition to these findings, a multivariate study testing the relationship of blood pressure to these enzyme activities, as well as to plasma levels of angiotensin I and renin activity, suggested abnormalities in the networks of proteolytic enzymes and in the peptide metabolism surrounding the renin-angiotensin system in SHR. These abnormalities may play some important roles in pathophysiological mechanisms of hypertension in SHR.     

Monitoring Spawning Activity in a Southern California Marine Protected Area Using Molecular Identification of Fish Eggs

In order to protect the diverse ecosystems of coastal California, a series of marine protected areas (MPAs) have been established. The ability of these MPAs to preserve and potentially enhance marine resources can only be assessed if these habitats are monitored through time. This study establishes a baseline for monitoring the spawning activity of fish in the MPAs adjacent to Scripps Institution of Oceanography (La Jolla, CA, USA) by sampling fish eggs from the plankton. Using vertical plankton net tows, 266 collections were made from the Scripps Pier between 23 August 2012 and 28 August 2014; a total of 21,269 eggs were obtained. Eggs were identified using DNA barcoding: the COI or 16S rRNA gene was amplified from individual eggs and sequenced. All eggs that were successfully sequenced could be identified from a database of molecular barcodes of California fish species, resulting in species-level identification of 13,249 eggs. Additionally, a surface transport model of coastal circulation driven by current maps from high frequency radar was used to construct probability maps that estimate spawning locations that gave rise to the collected eggs. These maps indicated that currents usually come from the north but water parcels tend to be retained within the MPA; eggs sampled at the Scripps Pier have a high probability of having been spawned within the MPA. The surface transport model also suggests that although larvae have a high probability of being retained within the MPA, there is also significant spillover into nearby areas outside the MPA. This study provides an important baseline for addressing the extent to which spawning patterns of coastal California species may be affected by future changes in the ocean environment.     

AtNUDX6, an ADP-Ribose/NADH Pyrophosphohydrolase in Arabidopsis,Positively Regulates NPR1-Dependent Salicylic Acid Signaling

Here, we investigated the physiological role of Arabidopsis (Arabidopsis thaliana) AtNUDX6, the gene encoding ADP-ribose (Rib)/NADH pyrophosphohydrolase, using its overexpressor (Pro_35S:AtNUDX6) or disruptant (KO-nudx6). The level of NADH in Pro_35S:AtNUDX6 and KO-nudx6 plants was decreased and increased, respectively, compared with that of the control plants, while the level of ADP-Rib was not changed in either plant. The activity of pyrophosphohydrolase toward NADH was enhanced and reduced in the Pro_35S:AtNUDX6 and KO-nudx6 plants, respectively. The decrease in the activity of NADH pyrophosphohydrolase and the increase in the level of NADH were observed in the rosette and cauline leaves, but not in the roots, of the KO-nudx6 plants. Notably, the expression level of AtNUDX6 and the activity of NADH pyrophosphohydrolase in the control plants, but not in the KO-nudx6 plants, were increased by the treatment with salicylic acid (SA). The expression of SA-induced genes (PR1, WRKY70, NIMIN1, and NIMIN2) depending on NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key component required for pathogen resistance, was significantly suppressed and enhanced in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively, under the treatment with SA. Induction of thioredoxin h5 (TRX-h5) expression, which catalyzes a SA-induced NPR1 activation, was suppressed and accelerated in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively. The expression of isochorismate synthase1, required for the regulation of SA synthesis through the NPR1-mediated feedback loop, was decreased and increased in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively. Judging from seed germination rates, the KO-nudx6 plants had enhanced sensitivity to the toxicity of high-level SA. These results indicated that AtNUDX6 is a modulator of NADH rather than ADP-Rib metabolism and that, through induction of TRX-h5 expression, AtNUDX6 significantly impacts the plant immune response as a positive regulator of NPR1-dependent SA signaling pathways.Nudix (nucleoside diphosphates linked to some moiety X) hydrolases are a phylogenetically widespread enzyme family and are widely distributed among all classes of organisms, such as bacteria, yeast, algae, nematodes, vertebrates, and plants (Bessman et al., 1996; Xu et al., 2004; Kraszewska, 2008). The enzymes catalyze, with varying degrees of substrate specificity, the hydrolysis of a variety of nucleoside diphosphate derivatives: nucleoside diphosphates and triphosphates and their oxidized forms, dinucleoside polyphosphates, nucleotide sugars, NADH, CoA, and the mRNA caps (McLennan, 2006; Kraszewska, 2008; Gunawardana et al., 2009). Since these compounds are often toxic to cells, Nudix hydrolases seem to play protective, regulatory, and signaling roles in metabolism by hydrolytically removing such compounds (Bessman et al., 1996; Xu et al., 2004).We reported the molecular and enzymatic characteristics of Nudix hydrolases (AtNUDX1–AtNUDX27) in Arabidopsis (Arabidopsis thaliana) plants (Ogawa et al., 2005, 2008). Notably, among 27 types of AtNUDXs, cytosolic AtNUDX2, AtNUDX6, AtNUDX7, and AtNUDX10 had pyrophosphohydrolase activity toward both ADP-Rib and NADH in vitro. Recent studies have shown that the actions of NADH and/or ADP-Rib pyrophosphohydrolases are closely related to defense systems in response to biotic and abiotic stresses in higher plants.It has been reported that the expression of AtNUDX7 is induced by avirulent pathogenic attacks. Knockout AtNUDX7 mutants (KO-nudx7) showed enhanced resistance against both virulent and avirulent bacterial strains (Bartsch et al., 2006; Jambunathan and Mahalingam, 2006; Adams-Phillips et al., 2008). In addition, it was revealed that AtNUDX7 functions as a negative regulator on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) signaling required for basal resistance to invasive pathogens (Bartsch et al., 2006); EDS1 regulates accumulation of the phenolic defense molecule, salicylic acid (SA), and other as yet unidentified signal intermediates and controls the defense activation and programmed cell death by collaborating with its interaction partner PHYTOALEXIN-DEFICIENT4 in cells surrounding pathogen infection foci. Furthermore, Ge et al. (2007) reported that AtNUDX7 functions to prevent excessive stimulation of the defense response, which is dependent on and independent of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a master regulator of SA-induced defense genes (SAIGs), and SA accumulation.On the other hand, we recently demonstrated the roles of Arabidopsis NADH/ADP-Rib pyrophosphohydrolases (AtNUDX2 and AtNUDX7) in tolerance to oxidative stress using the respective overexpressors (Pro_35S:AtNUDX2 and Pro_35S:AtNUDX7) or disruptants (KO-nudx7; Ishikawa et al., 2009; Ogawa et al., 2009). Interestingly, overexpression of AtNUDX2 and AtNUDX7 in Arabidopsis plants was responsible for an enhanced tolerance to oxidative stress derived from the treatment with paraquat (an agent producing O₂⁻) and salinity. Taken together, these results revealed that both AtNUDX2 and AtNUDX7 function in accelerating nucleotide recycling from ADP-Rib produced by poly(ADP-Rib) metabolism, leading to suppression of the overconsumption of NAD⁺ and ATP in Arabidopsis cells under stressful conditions. In addition, AtNUDX7 served to balance between NADH and NAD⁺ by NADH turnover and to regulate the defense mechanisms against DNA damage by modulation of the poly(ADP-ribosyl)ation (PAR) reaction through NADH metabolism in response to oxidative stress (Ishikawa et al., 2009; Ogawa et al., 2009). These findings clearly indicated that the regulation of NADH and/or ADP-Rib metabolism via Nudix hydrolases is involved in the responses to both biotic and abiotic stresses in higher plants.The question that we must consider next is whether the other AtNUDXs (AtNUDX6 and AtNUDX10) with pyrophosphohydrolase activities toward ADP-Rib and NADH are involved in the defense systems against oxidative stress and pathogen attack. The expression of AtNUDX6 has been reported to be induced by pathogenic attacks and treatment with the SA analogs 2,6-dichloroisonicotinic acid and acibenzolar-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH; Bartsch et al., 2006; Qiu et al., 2008; Knoth et al., 2009). Furthermore, the expression of AtNUDX6 was strongly dependent on EDS1 (Bartsch et al., 2006). However, the functional significance of AtNUDX6 is still unclear, since a loss-of-function mutant of AtNUDX6 has not yet been found.In this paper, to assess the physiological function of AtNUDX6, we identified an Arabidopsis mutant in which T-DNA is inserted into AtNUDX6 and subsequently studied the levels of ADP-Rib and NAD(H), PAR activity, expression of genes related to SA signaling, and SA tolerance in the AtNUDX6 overexpressors and disruptants in comparison with the AtNUDX7 disruptants. The results obtained here indicated that AtNUDX6 positively regulates NPR1-dependent SA signaling via modulation of NADH metabolism in the plant immune response.     

Direct observation of molecular motility by light microscopy

We used video-fluorescence microscopy to directly observe the sliding movement of single fluorescently labeled actin filaments along myosin fixed on a glass surface. Single actin filaments labeled with phalloidin-tetramethyl-rhodamine, which stabilizes the filament structure of actin, could be seen very clearly and continuously for at least 60 min in 02-free solution, and the sensitivity was high enough to see very short actin filaments less than 40 nm long that contained less than eight dye molecules. The actin filaments were observed to move along double-headed and, similarly, single-headed myosin filaments on which the density of the heads varied widely in the presence of ATP, showing that the cooperative interaction between the two heads of the myosin molecule is not essential to produce the sliding movement. The velocity of actin filament independent of filament length (greater than 1 micron) was almost unchanged until the density of myosin heads along the thick filament was decreased from six heads/14.3 nm to 1 head/34 nm. This result suggests that five to ten heads are sufficient to support the maximum sliding velocity of actin filaments (5 micron/s) under unloaded conditions. In order for five to ten myosin heads to achieve the observed maximum velocity, the sliding distance of actin filaments during one ATP cycle must be more than 60 nm.