Chiisanoside is not absorbed but inhibits oil absorption in the small intestine of rodents

Chiisanoside is the main component of Acanthopanax sessiliflorus leaves. Simultaneous administration of chiisanoside resulted in a decrease in the plasma TG level and increase of undigested TG in the intestinal lumen after oil gavage to mice. This suggests that chiisanoside has the potential to prevent obesity as a lipase inhibitor which suppresses fat absorption in vivo.     

Potential Myocardial Protection of 3,4-seco-Lupane Triterpenoids from Acanthopanax sessiliflorus Leaves

A rich of 3,4-seco-lupane triterpenoids including chiisanoside (CSS), divaroside (DVS), sessiloside-A1 (SSA) and chiisanogenin (CSG) were isolated from the ethanol extract of the leaves of Acanthopanax sessiliflorus. On the basis of previous studies, this article focused on four important components of 3,4-seco-lupane triterpenoids in Acanthopanax sessiliflorus leaves and explored their protective effects against aconitine-induced cardiomyocyte injury and their molecular mechanisms. The results showed that pretreatment with 3,4-seco-lupane triterpenoids could effectively increase cell viability, reduce CK-MB and LDH activities, reduce ROS production, maintain calcium concentration balance, and inhibit apoptosis, with divaroside having the best effect. In addition, Western blot results showed that divaroside down-regulated Cleaved caspase-3 and Bax and up-regulated Bcl-2 expression through activating the PI3 K/AKT pathway. However, the LY294002 inhibitor reversed this situation. This suggests that 3,4-seco-lupane triterpenoids may be a new hotspot for potential myocardial protective drugs research.     

In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase

We have previously shown that, in vivo, the integration system based on the gene encoding the TG1 integrase and the corresponding attB _TG1 and attP _TG1 sites works well not only in Streptomyces strains, but also in Escherichia coli. Furthermore, the attachment sites for TG1 integrase are distinct from those of ϕC31 integrase. In this report, we expressed TG1 integrase as a GST-TG1 integrase fusion protein and then used affinity separation and specific cleavage to release purified integrase. Conditions for in vitro recombination were established using the purified TG1 integrase and its cognate attP _TG1 and attB _TG1 sites. TG1 integrase efficiently catalyzed a site-specific recombination between attB _TG1 and attP _TG1 sites irrespective of their substrate topology. The minimal sequences of attP _TG1 and attB _TG1 sites required for the substrates of TG1 integrase were demonstrated to be 43 and 39-bp, respectively. These results provide the basic features of the TG1 integrase system to be used as biotechnological tools, as well as to unravel the mechanism of the serine integrase.     

Site-specific genome integration in alphaproteobacteria mediated by TG1 integrase

The serine-type phage integrase is an enzyme that catalyzes site-specific recombination between two attachment sites of phage and host bacterial genomes (attP and attB, respectively) having relatively short but distinct sequences without host auxiliary factor(s). Previously, we have established in vivo and in vitro site-specific recombination systems based on the serine-type integrase produced by actinophage TG1 and determined the minimal sizes of attP _TG1 and attB _TG1 sites required for the in vitro TG1 integrase reaction as 43- and 39-bp, respectively. Here, DNA databases were surveyed by FASTA program with the authentic attB _TG1 sequence of Streptomyces avermitilis as a query. As a result, possible attB _TG1 sequences were extracted from genomes of bacterial strains belonging to Class Alphaproteobacteria in addition to those of Class Actinobacteria. Those sequences extracted with a high similarity score and high sequence identity (we took arbitrarily more than 80% identity) turned out to be located within a conserved region of dapC or related genes encoding aminotransferases and proved to be actually recognized as the cognate substrate of attP _TG1 site by the in vitro TG1 integrase assay. Furthermore, the possible attB _TG1 site of Rhodospirillum rubrum revealed to be used actually as a native (endogenous) attachment site for the in vivo TG1-based integration system. These features are distinct from other serine-type phage integrases and advantageous for a tool of genome technology in varied industrially important bacteria belonging to Class Alphaproteobacteria.     

Protective Effects of 3,4-Seco-Lupane Triterpenes from Food Raw Materials of the Leaves of Eleutherococcus Senticosus and Eleutherococcus Sessiliflorus on Arrhythmia Induced by Barium Chloride

As a traditional wild vegetable and food raw material, the leaves of Eleutherococcus senticosus and Eleutherococcus sessiliflorus are rich in 3,4-seco-lupane triterpenes, including chiisanoside (CSS), divaroside (DVS), sessiloside-A (SSA), and chiisanogenin (CSG). This study was conducted to evaluate the anti-arrhythmic effects of these 3,4-seco-lupane triterpenes. Evaluation of the cytotoxicity of compounds was performed by measuring cell viability and apoptosis with the CCK-8 assay. In vivo, arrhythmia was induced by rapid injection of BaCl₂ via rat caudal vein. The occurrence time and duration of arrhythmias in rats were studied. The levels of SOD and MDA in serum, and Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in myocardial homogenate were detected by ELISA. The histopathological changes of rats myocardial were observed by HE staining. Changes in the expression of PKA and related proteins were detected by Western blot. The 3,4-seco-lupane triterpenes interactions with protein kinase A were analyzed by molecular docking. In the present study, we found that 3,4-seco-lupane triterpenes exhibited powerful anti-arrhythmic activity, especially DVS completely relieved the ventricular arrhythmia induced by BaCl₂. This study suggests that the leaves of E. senticosus and E. sessiliflorus might be used as functional food materials to prevent arrhythmia, and DVS can potentially be further developed as an anti-arrhythmic drug.     

Correlation of genetic and physical structure in the region surrounding the I2Fusarium oxysporum resistance locus in tomato

Summary The dominant gene I ₂ confers on tomato (Lycopersicon esculentum) resistance against the fungus Fusarium oxysporum f. sp. lycopersici race 2. A restriction fragment length polymorphism (RFLP) marker, TG105, has recently been found to be tightly linked to I ₂. The potential for cloning this gene by a reverse genetics approach prompted us to describe in both genetic and physical detail the region surrounding the I ₂ locus on chromosome 11. We have analyzed patterns of segregation of RFLP markers on chromosome 11 and Fusarium resistance in 140 F₂ plants from a cross between Fusarium-resistant and susceptible parental lines. Marker TG105 mapped 0.4 centi-Morgan (CM) from I ₂. Physical analysis of TG105 and its flanking RFLP markers, TG26 and TG36, by pulsed field gradient gel electrophoresis (PFGE) yielded a restriction map for this region encompassing at least 620 kb of the tomato genome. TG105 and TG26 hybridized to the same 175 kb MluI-NruI restriction fragment. We have therefore linked two genetically distinct RFLP markers. Based on the 4.1 cM distance between them, we have assigned a mean value of 43 kb for each cM recombination distance in the vicinity of I ₂. This local ratio between physical and genetic distances is more than 10-fold below the average for the tomato genome. It should therefore be possible to clone I ₂ by chromosome walking from TG105.     

Hyaluronidase and collagenase inhibitory activities of the herbal formulation <Emphasis Type="Italic">Triphala guggulu</Emphasis>

Myrrh (guggulu) oleoresin from the Commiphora mukul tree is an important component of antiarthritic drugs in Ayurvedic medicine. Clinical data suggest that elevated levels of hyaluronidase and collagenase type 2 enzymes contribute significantly to cartilage degradation. Triphala guggulu (TG) is a guggulu-based formulation used for the treatment of arthritis. We assessed the chondroprotective potential of TG by examining its effects on the activities of pure hyaluronidase and collagenase type 2 enzymes. Triphala shodith guggulu (TSG), an intermediate in the production of TG, was also examined. A spectrophotometric method was used to assay Hyaluronidase activity, and to detect potential Hyaluronidase inhibitors. Aqueous and hydro-alcoholic extracts of TSG showed weak but dose-dependent inhibition of hyaluronidase activity. In contrast, the TG formulation was 50 times more potent than the TSG extract with respect to hyaluronidase inhibitory activity. A validated X-ray film-based assay was used to measure the gelatinase activity of pure collagenase type 2. Hydro-alcoholic extracts of the TG formulation were 4 times more potent than TSG with respect to collagenase inhibitory activity. Components of Triphala were also evaluated for their inhibitory activities on hyaluronidase and collagenase. This is the first report to show that the T2 component of Triphala (T. chebula) is a highly potent hyaluronidase and collagenase inhibitor. Thus, the TG formulation inhibits two major enzymes that can degrade cartilage matrix. Our study provides the first in vitro preclinical evidence of the chondroprotective properties of TG.     

Fine mapping of three quantitative trait loci for late blight resistance in tomato using near isogenic lines (NILs) and sub-NILs

An earlier study identified quantitative trait loci (QTLs) lb4, lb5b, and lb11b for quantitative resistance to Phytophthora infestans (late blight) in a backcross population derived from crossing susceptible cultivated tomato (Lycopersicon esculentum) with resistant L. hirsutum. The QTLs were located in intervals spanning 28–47 cM. Subsequently, near-isogenic lines (NILs) were developed for lb4, lb5b, and lb11b by marker-assisted backcrossing to L. esculentum. Sub-NILs containing overlapping L. hirsutum segments across each QTL region were selected and used to validate the QTL effects, fine-map QTLs, and evaluate potential linkage drag between resistance QTLs and QTLs for horticultural traits. The NILs and sub-NILs were evaluated for disease resistance and eight horticultural traits at three field locations. Resistance QTLs were detected in all three sets of NIL lines, confirming the BC₁ mapping results. Lb4 mapped near TG609, and between TG182 and CT194, on chromosome 4, a 6.9-cM interval; lb5b mapped to an 8.8-cM interval between TG69a and TG413 on chromosome 5, with the most likely position near TG23; and lb11b mapped to a 15.1-cM interval on chromosome 11 between TG194 and TG400, with the peak centered between CT182 and TG147. Most QTLs for horticultural traits were identified in intervals adjacent to those containing the late blight resistance QTLs. Fine mapping of these QTLs permits the use of marker-assisted selection for the precise introgression of L. hirsutum segments containing late blight resistance alleles separately from those containing deleterious alleles at horticulturally important QTLs.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by D.B. Neale     

Null mutations of NT-3 and Bax affect trigeminal ganglion cell number but not brainstem barrelette pattern formation

Abstract

Trigeminal ganglion (TG) neurons innervate the grid-like array of whisker follicles on the face of the mouse. Central TG axons project to the trigeminal (V) brainstem nuclear complex, including the nucleus principalis (PrV) and the spinal subnucleus interpolaris (SpVi), where they innervate barrelettes that are organized in a pattern that recapitulates the whisker pattern on the face. Neurotrophin-3 (NT-3) supports a population of TG cells that supply slowly adapting mechanoreceptors in the whisker pad. We examined mice at embryonic day 17 (E17) and on the day of birth (P0) with null mutations of NT-3, Bax, a proapoptotic gene associated with naturally occurring cell death, and Bax/NT-3 double knockout (KO) mutants to determine if: (1) the number of TG cells would be reduced; (2) eliminating the Bax gene would rescue the NT-3-dependent neurons; and (3) the central projections of the rescued axons in the Bax/NT-3 double KO mice would fail to develop the barrelette patterns in the PrV and SpVi subnuclei. In mice at E17, NT-3^?/? mutants had 65% fewer TG neurons than found in age-matched wild-type (WT) mice, and at P0, the number was reduced by 55% (p?<?0.001 for both). Bax null mutant mice at E17 had 132% of the WT number of TG cells (p?<?0.001), although the numbers returned to WT levels by P0. Bax/NT-3 double KO mice at E17 had TG cell numbers equal to those seen in WT, but the double KO failed to retain WT TG neuron numbers in P0 mice (39% fewer cells; p?<?0.001). In all cases of reduced experimental neuron numbers, and in the E17 Bax^?/? mice with supernumerary cells, the barrelette patterns in the PrV and SpVi were normal. Only a slight qualitative reduction in overall barrelette field area and clarity of barrelettes were seen. These results suggest that NT-3 is not necessary for barrelette pattern formation in the brainstem.     

Effects of the FABP2 A54T Mutation on Triglyceride Metabolism of Viscerally Obese Men

Objective: Viscerally obese individuals are frequently characterized by a proatherogenic condition. A missense mutation (A54T) in the fatty acid binding protein type 2 (FABP2) gene has been associated with insulin resistance and obesity. This study examined the effect of this mutation on lipoprotein levels in viscerally obese hyperinsulinemic condition. Research Methods and Procedures: A total of 217 men were assigned to one of two groups based on their FABP2 A54T polymorphism. Results: The two genotypic groups showed no difference in either physiological characteristics or lipoprotein/lipid profile, before or after statistical adjustment for age. From this initial sample, 50 men accepted to have their postprandial lipid response assessed and 10 T54/A54 heterozygotes were then individually matched for visceral adipose tissue accumulation and fasting plasma triglyceride (TG) levels with 10 A54/A54 homozygotes. High‐density lipoprotein (HDL)‐TG levels were significantly increased in the fasting state as well as 4 hours after the test meal (p = 0.04 and p = 0.0008, respectively) in men bearing the A54T mutation. In addition, the area under the curve of postprandial HDL‐TG levels was also significantly higher among T54/A54 heterozygotes than among A54/A54 homozygotes (p = 0.04). Interestingly, fasting TG concentrations in large TG‐rich lipoproteins (large‐TRL; S_f > 400) were correlated with HDL‐TG levels at 4 (r = 0.74, p = 0.01) and 8 hours (r = 0.73, p = 0.01) after the test meal in T54/A54 heterozygotes only. Discussion: The FABP2 A54T missense mutation may contribute to the TG enrichment of HDL in the postprandial state that, in turn, may alter the risk of atherosclerotic vascular disease.     

A Triglyceride‐Raising APOA5 Genetic Variant Is Negatively Associated With Obesity and BMI in the Chinese Population

Apolipoprotein A‐V (apo A‐V) exerts a potent triglyceride (TG)‐lowering effect through enhanced intravascular TG‐hydrolysis with increased uptake of TG‐derived free fatty acids into muscle and adipose tissue. Genetic variants in the APOA5 gene were strongly associated with plasma TG concentrations. The aim of this study was to examine whether APOA5 genetic variation was associated with obesity. We genotyped the missense c.553 G>T polymorphism (p.G185C) in the APOA5 gene in 1,085 Chinese (333 obese subjects and 752 nonobese controls). We analyzed the association between the c.553 G>T polymorphism and obesity and related metabolic phenotypes. The T allele at the c.553 G>T polymorphism was associated with higher plasma TG concentrations. Each additional T allele was associated with an increased TG concentration of 53.5 mg/dl (95% confidence interval (CI) 29.6–76.0, P < 0.0001). However, the T allele was associated lower risk of obesity (odds ratio (OR), 0.48; 95% CI 0.32–0.73, P = 0.0004). Each additional copy of the T allele was associated with a BMI decrease of 0.73 kg/m² (95% CI 0.26–1.16, P = 0.002), equivalent to 2.11 kg in a person 1.7 m tall. We may then conclude that the TG‐raising APOA5 genetic variant was associated with a decrease in BMI and reduced risk of obesity in the Chinese population.     

Anti‐lipolytic Effects of Insulin in African American and White Prepubertal Boys

Objective: Relative to whites, African Americans have lower circulating triglycerides (TG) and greater highdensity lipoprotein cholesterol. The metabolic basis for this difference is not known. This study was conducted to test the hypothesis that insulin‐induced suppression of free fatty acids (FFA) results in lower serum TG in African American versus white prepubertal children. Research Methods and Procedures: Insulin, FFA, and TG were determined at baseline and during a frequently sampled, intravenous glucose tolerance test in eight African American and eight white prepubertal males pair‐matched for whole‐body insulin sensitivity. Results: Baseline TG was lower in African Americans (0.43 ± 0.10 vs. 0.79 ± 0.37 mM/L; mean ± SD; p < 0.01). African Americans had higher peak insulin (218 ± 102 vs. 100 ± 30 pM/L; mean ± SD; p < 0.01) and a greater acute insulin response (9282 ± 4272 vs. 4230 ± 1326 pM/L × 10 minutes; mean ± SD; p < 0.05). FFA and TG values determined at the FFA nadir were lower in African Americans (0.26 ± 0.02 vs. 0.30 ± 0.03 mEq/L; mean ± SD; p < 0.01 for FFA nadir and 0.49 ± 0.07 vs. 0.77 ± 0.33 mM/L; mean ± SD; p < 0.05 for TG). Among all subjects, FFA nadir was correlated with peak insulin (r = ?0.54; p < 0.05). After adjusting for FFA nadir, neither baseline nor postchallenge TG differed with ethnicity (p = 0.073 and 0.192, respectively). The ethnic difference in FFA nadir disappeared after adjusting for peak insulin (p = 0.073). Discussion: These data suggest that hyperinsulinemiainduced suppression of FFA among African Americans is a determinant of lower TG in this group.     

Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc_n, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc_n. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.     

Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury

Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2^+/? mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.     

Visualisation of transglutaminase-mediated cross-linking activity in germinating pollen by laser confocal microscopy

Abstract

Transglutaminases (TGs) are a multigenic family of calcium-dependent protein cross-linking enzymes, which are present in animal and plant cells. We have previously reported the presence of TGs in the cytosol and, more recently, in the cell wall of Malus domestica pollen, where it may be involved in pollen germination and pollen–stylar interactions. In this report we describe a simple method for the in situ visualisation of TG activity in germinating pollen. The method is based on the incorporation, mediated by pollen TG, of a fluorescently labelled exogenous diamine substrate of TG (fluorescein-cadaverine) into endogenous pollen substrates. Following the in situ TG activity reaction, the presence of cross-linked pollen proteins was visualised in fixed specimens of germinated pollen by laser confocal microscopy. Our data indicate the presence of TG cross-linking activity mainly at the apical part of the pollen tube, in the region proximal to the grain, and in the pollen grain itself. In planta, the products of this activity may provide strength to the pollen tube migrating through the style.     

Glucokinase regulatory protein gene polymorphism affects postprandial lipemic response in a dietary intervention study

Postprandial triglyceridemia is an emerging risk factor for cardiovascular disease. However, most of the genes that influence postprandial triglyceridemia are not known. We evaluated whether a common nonsynonymous SNP rs1260326/P446L in the glucokinase regulatory protein (GCKR) gene influenced variation in the postprandial lipid response after a high-fat challenge in seven hundred and seventy participants in the Amish HAPI Heart Study who underwent an oral high-fat challenge and had blood samples taken in the fasting state and during the postprandial phase at 1, 2, 3, 4, and 6 h. We found that the minor T allele at rs1260326 was associated with significantly higher fasting TG levels after adjusting for age, sex, and family structure (P _a = 0.06 for additive model, and P _r = 0.0003 for recessive model). During the fat challenge, the T allele was associated with significantly higher maximum TG level (P _a = 0.006), incremental maximum TG level (P _a = 0.006), TG area under the curve (P _a = 0.02) and incremental TG area under the curve (P _a = 0.03). Our data indicate that the rs1260326 T allele of GCKR is associated with both higher fasting levels of TG as well as the postprandial TG response, which may result in higher atherogenic risk.     

Regulation of transglutaminase 1 gene expression by 12-O-tetradecanoylphorbol-13-acetate, dexamethasone, and retinoic acid in cultured human keratinocytes

Transglutaminase 1 (TG1) is an enzyme that is expressed at the late stage of terminal differentiation of keratinocytes and catalyzes the ε-(γ-glutamyl)lysine cross-linking reaction to form a highly insoluble cell envelope. To elucidate the mechanism of TG1 gene expression in keratinocytes, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), dexamethasone, 1,25-dihydroxyvitamin D₃, and retinoic acid on the levels of TG1 mRNA in cultured normal human epidermal keratinocytes (NHEK). Treatment of NHEK with TPA, Up to 10 nM, markedly increased the levels of TG1 mRNA in a dose-dependent manner. The effect by treatment with 1 nM TPA reached a peak after 16 h of incubation (20-fold above the basal level). In contrast, phorbol had no effect on TG1 gene expression. The induction of TG1 mRNA expression by TPA was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine. Dexamethasone at a concentration of 1 μM also increased the TG1 mRNA levels, but the maximum induction was observed (3-fold above the basal level) after 72 h of incubation. The effect of dexamethasone was not suppressed by H-7. Moreover, 1 μM of retinoic acid completely inhibited the induction of TG1 mRNA by both TPA and dexamethasone. 1,25-Dihydroxyvitamin D₃ showed no effect on the TG1 mRNA levels. From these results, we suggest that the expression of TG1 gene may be upregulated by protein kinase C and glucocorticoid receptor systems and down-regulated by the retinoic acid receptor system.     

Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing

Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single‐nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild‐type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype.     

Heterologous expression of Arabidopsis H+‐pyrophosphatase enhances salt tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.)

The Arabidopsis vacuolar H⁺‐pyrophosphatase (AVP1), when over‐expressed in transgenic (TG) plants, regulates root and shoot development via facilitation of auxin flux, and enhances plant resistance to salt and drought stresses. Here, we report that TG perennial creeping bentgrass plants over‐expressing AVP1 exhibited improved resistance to salinity than wild‐type (WT) controls. Compared to WT plants, TGs grew well in the presence of 100 mm NaCl, and exhibited higher tolerance and faster recovery from damages from exposure to 200 and 300 mm NaCl. The improved performance of the TG plants was associated with higher relative water content (RWC), higher Na⁺ uptake and lower solute leakage in leaf tissues, and with higher concentrations of Na⁺, K⁺, Cl^‐ and total phosphorus in root tissues. Under salt stress, proline content was increased in both WT and TG plants, but more significantly in TGs. Moreover, TG plants exhibited greater biomass production than WT controls under both normal and elevated salinity conditions. When subjected to salt stress, fresh (FW) and dry weights (DW) of both leaves and roots decreased more significantly in WT than in TG plants. Our results demonstrated the great potential of genetic manipulation of vacuolar H⁺‐pyrophosphatase expression in TG perennial species for improvement of plant abiotic stress resistance.     

Thapsigargin,a selective inhibitor of sarco-endoplasmic reticulum Ca<Superscript>2+</Superscript>-ATPases,modulates nitric oxide production and cell death of primary rat hepatocytes in culture

Increased cytosolic calcium ([Ca²⁺] _i ) and nitric oxide (NO) are suggested to be associated with apoptosis that is a main feature of many liver diseases and is characterized by biochemical and morphological features. We sought to investigate the events of increase in [Ca²⁺] _i and endoplasmic reticulum (ER) calcium depletion by thapsigargin (TG), a selective inhibitor of sarco-ER-Ca²⁺-ATPases, in relation to NO production and apoptotic and necrotic markers of cell death in primary rat hepatocyte culture. Cultured hepatocytes were treated with TG (1 and 5 μmol/L) for 0–24 or 24–48 h. NO production and inducible NO synthase (iNOS) expression were determined as nitrite levels and by iNOS-specific antibody, respectively. Hepatocyte apoptosis was estimated by caspase-3 activity, cytosolic cytochrome c content and DNA fragmentation, and morphologically using Annexin-V/propidium iodide staining. Hepatocyte viability and mitochondrial activity were evaluated by ALT leakage and MTT test. Increasing basal [Ca²⁺] _i by TG, NO production and apoptotic/necrotic parameters were altered in different ways, depending on TG concentration and incubation time. During 0–24 h, TG dose-dependently decreased iNOS-mediated spontaneous NO production and simultaneously enhanced hepatocyte apoptosis. In addition, TG 5 μmol/L produced secondary necrosis. During 24–48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 μmol/L further promoted mitochondrial damage as demonstrated by cytochrome c release. A selective iNOS inhibitor, aminoguanidine, suppressed TG-stimulated NO production and ALT leakage from hepatocytes after 24–48 h. Our data suggest that the extent of the [Ca²⁺] _i increase and the modulation of NO production due to TG treatment contribute to hepatocyte apoptotic and/or necrotic events.