Ammonium accumulation is associated with senescence of rice leaves

The relationship between ammonium accumulation and senescence of detached rice leaves was investigated. Ammonium accumulation in detached rice leaves coincided closely with dark-induced senescence. Exogenous NH4Cl and methionine sulfoximine, which caused an accumulation of ammonium in detached rice leaves, promoted senescence. Treatments such as light and benzyladenine, which retarded senescence, decreased ammonium level in detached rice leaves. Abscisic acid, which promoted senescence, increased ammonium level in detached rice leaves. The current results suggest that ammonium accumulation may be involved in regulating senescence. Evidence was presented to show that ammonium accumulated in detached rice leaves increases tissue sensitivity to ethylene. The accumulation of ammonium in detached rice leaves during dark-induced senescence is attributed to a decrease in glutamine synthetase activity and an increase in reduction of nitrate.     

Colchicine modulates calcium homeostasis and electrical property of HL‐1 cells

Colchicine is a microtubule disruptor that reduces the occurrence of atrial fibrillation (AF) after an operation or ablation. However, knowledge of the effects of colchicine on atrial myocytes is limited. The aim of this study was to determine if colchicine can regulate calcium (Ca²⁺) homeostasis and attenuate the electrical effects of the extracellular matrix on atrial myocytes. Whole‐cell clamp, confocal microscopy with fluorescence, and western blotting were used to evaluate the action potential and ionic currents of HL‐1 cells treated with and without (control) colchicine (3 nM) for 24 hrs. Compared with control cells, colchicine‐treated HL‐1 cells had a longer action potential duration with smaller intracellular Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ content by 10% and 47%, respectively. Colchicine‐treated HL‐1 cells showed a smaller L‐type Ca²⁺ current, reverse mode sodium–calcium exchanger (NCX) current and transient outward potassium current than control cells, but had a similar ultra‐rapid activating outward potassium current and apamin‐sensitive small‐conductance Ca²⁺‐activated potassium current compared with control cells. Colchicine‐treated HL‐1 cells expressed less SERCA2a, total, Thr17‐phosphorylated phospholamban, Cav1.2, CaMKII, NCX, Kv1.4 and Kv1.5, but they expressed similar levels of the ryanodine receptor, Ser16‐phosphorylated phospholamban and Kv4.2. Colchicine attenuated the shortening of the collagen‐induced action potential duration in HL‐1 cells. These findings suggest that colchicine modulates the atrial electrical activity and Ca²⁺ regulation and attenuates the electrical effects of collagen, which may contribute to its anti‐AF activity.     

Sodium and Calcium Inward Currents in Freshly Dissociated Smooth Myocytes of Rat Uterus

Freshly dissociated myocytes from nonpregnant, pregnant, and postpartum rat uteri have been studied with the tight-seal patch-clamp method. The inward current contains both I_Na and I_Ca that are vastly different from those in tissue-cultured material. I_Na is abolished by Na⁺-free medium and by 1 μM tetrodotoxin. It first appears at ∼−40 mV, reaches maximum at 0 mV, and reverses at 84 mV. It activates with a voltage-dependent τ of 0.2 ms at 20 mV, and inactivates as a single exponential with a τ of 0.4 ms. Na⁺ conductance is half activated at −21.5 mV, and half inactivated at −59 mV. I_Na reactivates with a τ of 20 ms. I_Ca is abolished by Ca²⁺-free medium, Co²⁺ (5 mM), or nisoldipine (2 μM), and enhanced in 30 mM Ca²⁺, Ba²⁺, or BAY-K 8644. It first appears at ∼−30 mV and reaches maximum at +10 mV. It activates with a voltage-dependent τ of 1.5 ms at 20 mV, and inactivates in two exponential phases, with τ''s of 33 and 133 ms. Ca²⁺ conductance is half activated at −7.4 mV, and half inactivated at −34 mV. I_Ca reactivates with τ''s of 27 and 374 ms. I_Na and I_Ca are seen in myocytes from nonpregnant estrus uteri and throughout pregnancy, exhibiting complex changes. The ratio of densities of peak I_Na/I_Ca changes from 0.5 in the nonpregnant state to 1.6 at term. The enhanced role of I_Na, with faster kinetics, allows more frequent repetitive spike discharges to facilitate simultaneous excitation of the parturient uterus. In postpartum, both currents decrease markedly, with I_Na vanishing from most myocytes. Estrogen-enhanced genomic influences may account for the emergence of I_Na, and increased densities of I_Na and I_Ca as pregnancy progresses. Other influences may regulate varied channel expression at different stages of pregnancy.     

Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases’ cascade

Antizymes delicately regulate ornithine decarboxylase (ODC) enzyme activity and polyamine transportation. One member of the family, antizyme-1, plays vital roles in molecular and cellular functions, including developmental regulation, cell cycle, proliferation, cell death, differentiation and tumorigenesis. However, the question of how does it participate in the cell apoptotic mechanism is still unsolved. To elucidate the contribution of human antizyme-1 in haematopoietic cell death, we examine whether inducible overexpression of antizyme enhances apoptotic cell death. Antizyme reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells, acute T leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G₁ appearance, loss of mitochondrial membrane potential (Δψ _m ), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following conditional antizyme overexpression, all protein levels of cyclin-dependent kinases (Cdks) and cyclins are not significantly reduced, except cyclin D, before their entrance into apoptotic cell death. However, introduced cyclin D1 into Jurkat T tetracycline (Tet)-On cell system still couldn’t rescue cells from apoptosis. Antizyme doesn’t influence the expression of tumor suppressor p53 and its downstream p21, but it interferes in the expressions of Bcl-2 family. Inducible antizyme largely enters mitochondria resulting in cytochrome c release from mitochondria to cytosol following Bcl-xL decrease and Bax increase. According to these data, we suggest that antizyme induces apoptosis mainly through mitochondria-mediated and cell cycle-independent pathway. Furthermore, antizyme induces apoptosis not only by Bax accumulation reducing the function of the Bcl-2 family, destroying the Δψ _m , and releasing cytochrome c to cytoplasm but also by the activation of apoptosomal caspase cascade.     

Molecular dissection of mitogillin reveals that the fungal ribotoxins are a family of natural genetically engineered ribonucleases

Mitogillin and the related fungal ribotoxins are highly specific ribonucleases which inactivate the ribosome enzymatically by cleaving the 23-28 S RNA of the large ribosomal subunit at a single phosphodiester bond. The site of cleavage occurs between G4325 and A4326 (rat ribosome numbering) which are present in one of the most conserved sequences (the alpha-sarcin loop) among the large subunit ribosomal RNAs of all living species. Amino acid sequence comparison of ribotoxins and guanyl/purine ribonucleases have identified domains or residues likely involved in ribonucleolytic activity or cleavage specificity. Fifteen deletion mutants (each 4 to 8 amino acid deletions) in motifs of mitogillin showing little amino acid sequence homology with guanyl/purine ribonucleases were constructed by site-directed mutagenesis. Analyses of the purified mutant proteins identified those regions in fungal ribotoxins contributing to ribosome targeting and modulating the catalytic activity of the toxin; some of the identified motifs are homologous to sequences in ribosomal proteins and elongation factors. This mutational study of mitogillin together with the recently published x-ray structure of restrictocin (a close relative of mitogillin) supports the hypothesis that the specific cleavage properties of ribotoxins are the result of natural genetic engineering in which the ribosomal targeting elements of ribosome-associated proteins were inserted into nonessential regions of T1-like ribonucleases.     

The responses of photosynthetic gas exchange and chlorophyll a fluorescence to changes of irradiance and temperature in two species of Miscanthus

Photosynthetic CO₂ uptake and chlorophyll (Chl) a fluorescence of C₄ perennial grasses, Miscanthus floridulus (Labill) Warb and M. transmorrisonensis Hayata, from altitudes in central Taiwan of 390 and 2700 m, respectively, were studied at 10 and 25 °C to find if the species differ in their photosynthetic responses to a low temperature, and whether their photosystems 2 become more susceptible to the photoinhibition at low temperatures. For both species, the maximum photosynthetic rate (P_max) was reduced when the leaves were exposed to 10 °C. At irradiances higher than 400 μmol m^-2 s^-1, the values of F_v/F_m were reduced in both species at 10 °C but not at 25 °C, which indicated the photoinhibition at 10 °C. Reductions in P_max and the values of F_v/F_m at 10 °C were lesser in M. transmorrisonensis than in M. floridulus. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism.

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.     

Distinguishing positive selection from neutral evolution: boosting the performance of summary statistics

Summary statistics are widely used in population genetics, but they suffer from the drawback that no simple sufficient summary statistic exists, which captures all information required to distinguish different evolutionary hypotheses. Here, we apply boosting, a recent statistical method that combines simple classification rules to maximize their joint predictive performance. We show that our implementation of boosting has a high power to detect selective sweeps. Demographic events, such as bottlenecks, do not result in a large excess of false positives. A comparison to other neutrality tests shows that our boosting implementation performs well compared to other neutrality tests. Furthermore, we evaluated the relative contribution of different summary statistics to the identification of selection and found that for recent sweeps integrated haplotype homozygosity is very informative whereas older sweeps are better detected by Tajima's π. Overall, Watterson's was found to contribute the most information for distinguishing between bottlenecks and selection.     

VPg-primed RNA synthesis of norovirus RNA-dependent RNA polymerases by using a novel cell-based assay

Molecular studies of human noroviruses (NoV) have been hampered by the lack of a permissive cell culture system. We have developed a sensitive and reliable mammalian cell-based assay for the human NoV GII.4 strain RNA-dependent RNA polymerase (RdRp). The assay is based on the finding that RNAs synthesized by transiently expressed RdRp can stimulate retinoic acid-inducible gene I (RIG-I)-dependent reporter luciferase production via the beta interferon promoter. Comparable activities were observed for the murine norovirus (MNV) RdRp. RdRps with mutations at divalent metal ion binding residues did not activate RIG-I signaling. Furthermore, both NoV and MNV RdRp activities were stimulated by the coexpression of their respective VPg proteins, while mutations in the putative site of nucleotide linkage on VPg abolished most of their stimulatory effects. Sequencing of the RNAs linked to VPg revealed that the cellular trans-Golgi network protein 2 (TGOLN2) mRNA was the template for VPg-primed RNA synthesis. Small interfering RNA knockdown of RNase L abolished the enhancement of signaling that occurred in the presence of VPg. Finally, the coexpression of each of the other NoV proteins revealed that p48 (also known as NS1-2) and VP1 enhanced and that VP2 reduced the RdRp activity. The assay should be useful for the dissection of the requirements for NoV RNA synthesis as well as the identification of inhibitors of the NoV RdRp.     

Increased Expression of Intranuclear Matrix Metalloproteinase 9 in Atrophic Renal Tubules Is Associated with Renal Fibrosis

Background

Reduced turnover of extracellular matrix has a role in renal fibrosis. Matrix metalloproteinases (MMPs) is associated with many glomerular diseases, but the histological association of MMPs and human renal fibrosis is unclear.

Methods

This is a retrospective study. Institutional Review Board approval was obtained for the review of patients’ medical records, data analysis and pathological specimens staining with waiver of informed consents. Specimens of forty-six patients were examined by immunohistochemical stain of MMP-9 in nephrectomized kidneys, and the association of renal expression of MMP-9 and renal fibrosis was determined. MMP-9 expression in individual renal components and fibrosis was graded as high or low based on MMP-9 staining and fibrotic scores.

Results

Patients with high interstitial fibrosis scores (IFS) and glomerular fibrosis scores (GFS) had significantly higher serum creatinine, lower estimated glomerular filtration rate (eGFR), and were more likely to have chronic kidney disease (CKD) and urothelial cell carcinoma. Univariate analysis showed that IFS and GFS were negatively associated with normal and atrophic tubular cytoplasmic MMP-9 expression and IFS was positively correlated with atrophic tubular nuclear MMP-9 expression. Multivariate stepwise regression indicated that MMP-9 expression in atrophic tubular nuclei (r = 0.4, p = 0.002) was an independent predictor of IFS, and that MMP-9 expression in normal tubular cytoplasm (r = −0.465, p<0.001) was an independent predictor of GFS.

Conclusions

Interstitial fibrosis correlated with MMP-9 expression in the atrophic tubular nuclei. Our results indicate that renal fibrosis is associated with a decline of MMP-9 expression in the cytoplasm of normal tubular cells and increased expression of MMP-9 in the nuclei of tubular atrophic renal tubules.