Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na+/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [3H]cytidine, but not [3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [3H]uridine, a typical substrate of NT. [3H]Cytidine and [3H]uridine were taken up via CNT2, in temperature-, extracellular Na+-, and substrate concentration-dependent manners. The uptake of [3H]cytidine and [3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [3H]uridine uptake by cytidine was competitive. The [3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.     

A single circularly permuted GFP sensor for inositol-1,3,4,5-tetrakisphosphate based on a split PH domain

A fluorescent sensor for the detection of inositol-1,3,4,5-tetrakisphosphate, Ins(1,3,4,5)P₄, was constructed from a split PH domain and a single circularly permuted GFP. A structure-based design was conducted to transduce a ligand-induced subtle structural perturbation of the split PH domain to an alteration in the population of the protonated and the deprotonated states of the GFP chromophore. Excitation of each distinct absorption band corresponding to the protonated or the deprotonated state of GFP resulted an increase and a decrease, respectively, in the intensity of emission spectra upon addition of Ins(1,3,4,5)P₄ to the split PH domain-based sensor. The Ins(1,3,4,5)P₄ sensor retained the ligand affinity and the selectivity of the parent PH domain, and realized the ratiometric fluorescence detection of Ins(1,3,4,5)P₄.     

Arachidonic acid inhibits Cl secretion in cultures of dog tracheal epithelium.

We studied the effects of arachidonic acid (AA) on Cl secretion across primary cultures of dog tracheal epithelium. Cell sheets showed mean values for baseline short-circuit current (Isc) and transepithelial resistance of 33.8 muA/cm2 and 360 omega.cm2 (n = 44). AA (5 x 10(-5) M) added to both sides increased Isc by 27.8 +/- 5.2 muA/cm2 (mean +/- SE, n = 8), and elevated intracellular cAMP levels. In the presence of 5 x 10(-6) M of both indomethacin (INDO) and nordihydroguaiaretic acid (NDGA) (inhibitors of cyclooxygenase and lipoxygenase, respectively), AA reduced Isc by 4.4 +/- 0.6 muA/cm2 (n = 10) without changing cAMP. Both INDO and NDGA were necessary to abolish the Isc increase in response to AA. The effects of AA on Isc were unaffected by amiloride. In the presence of INDO and NDGA, isoproterenol (ISO) raised cAMP and increased Isc by 27.6 +/- 4.3 (transient) and 12.8 +/- 3.2 muA/cm2 (sustained) (n = 9). With AA present as well as INDO and NDGA, the transient and sustained responses to ISO were significantly reduced to 13.2 +/- 2.4 and 3.9 +/- 0.8 muA/cm2 (n = 10), respectively; the increase in cAMP was unaltered. AA approximately halved baseline efflux of 125I from confluent cell sheets in high K medium and reduced the isoproterenol-induced increase in efflux to 20% of control. These data are consistent with the reported inhibitory effect of AA on apical membrane chloride channels.     

Mapping quantitative trait loci for root development under hypoxia conditions in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Key message

Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method.

Abstract

Waterlogging is a major environmental stress limiting soybean yield in wet parts of the world. Root development is an important indicator of hypoxia tolerance in soybean. However, little is known about the genetic control of root development under hypoxia. This study was conducted to identify quantitative trait loci (QTLs) responsible for root development under hypoxia. Recombinant inbred lines (RILs) developed from a cross between a hypoxia-sensitive cultivar, Tachinagaha, and a tolerant landrace, Iyodaizu, were used. Seedlings were subjected to hypoxia, and root development was evaluated with the value change in root traits between after and before treatments. We found 230 polymorphic markers spanning 2519.2 cM distributed on all 20 chromosomes (Chrs.). Using these, we found 11 QTLs for root length (RL), root length development (RLD), root surface area (RSA), root surface area development (RSAD), root diameter (RD), and change in average root diameter (CARD) on Chrs. 11, 12, 13 and 14, and 7 QTLs for hypoxia tolerance of these root traits. These included QTLs for RLD and RSAD between markers Satt052 and Satt302 on Chr. 12, which are important markers of hypoxia tolerance in soybean; those QTLs were stable between 2 years. To validate the QTLs, we developed a near-isogenic line with the QTL region derived from Iyodaizu. The line performed well under both hypoxia and waterlogging, suggesting that the region contains one or more genes with large effects on root development. These findings may be useful for fine mapping and positional cloning of gene responsible for root development under hypoxia.

     

Ability of the endangered species Papaver fauriei to produce hybrids with a cultivated poppy (Papaver sp.)

Papaver fauriei is an endemic and endangered species that grows only on the gravelly alpine slopes of Mt. Rishiri, Japan. Cultivated poppy (Papaver sp.), the species name of which is unknown, has been introduced to the natural habitats of P. fauriei through human activities. Because the appearance and internal transcribed spacer (ITS) sequences of these two poppies are highly similar, it is of concern that they could produce hybrids in their natural habitats. Thus, first, the ability of these two poppies to produce hybrids was analyzed by artificial fertilization in this study. A large number of seeds were produced by reciprocal crosses between P. fauriei and the cultivated poppy, comparable with the number of seeds obtained by self‐ or cross‐fertilization of P. fauriei or the cultivated poppy. In addition, high germination was observed for seeds obtained from crosses between the two poppies, and deleterious phenotypes, such as albinism and dwarfism, were not detected in the F₁ generation. These results indicate that after pollination, there is no reproductive isolation between the two poppies. Second, we sequenced the internal transcribed spacer (ITS) region of 240 poppy individuals collected from the gravelly alpine slopes of Mt. Rishiri, and 66 showed the sequence of P. fauriei, whereas 174 showed the sequence of the cultivated poppy. However, the ITS sequence that confirms hybridism between the two poppies was not detected in these individuals, indicating that hybridization of P. fauriei and the cultivated poppy rarely occurs under natural conditions. Unknown mechanism(s) appear to prevent cross‐pollination between the two poppies.     

The mitochondrial Ca2+ uptake regulator,MICU1, is involved in cold stress‐induced ferroptosis

Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia‐reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation‐dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome‐wide CRISPR screening, we found that a mitochondrial Ca²⁺ uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress‐induced ferroptosis. MICU1 is required for mitochondrial Ca²⁺ increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1‐dependent mitochondrial Ca²⁺ homeostasis‐MMP hyperpolarization axis is involved in cold stress‐induced lipid peroxidation and ferroptosis.