Search Results (9)

Accurate gene expression quantification using reverse transcription quantitative PCR (RT-qPCR) requires stable reference genes (RGs) for reliable normalization. However, few studies have systematically identified RGs suitable for simultaneous high salt, alkaline, and high-temperature conditions. This study addresses this gap by evaluating the stability of eight candidate RGs in the anaerobic halophilic alkalithermophile Natranaerobius thermophilus JW/NM-WN-LF^T under combined salt, alkali, and thermal stresses. The stability of these candidate RGs was assessed using five statistical algorithms: Delta CT, geNorm, NormFinder, BestKeeper, and RefFinder. Results indicated that recA exhibited the highest expression stability across all tested conditions and proved adequate as a single RG for normalization in both RT-qPCR and droplet digital PCR (ddPCR) assays. Furthermore, recA alone or combined with other RGs (sigA, rsmH) effectively normalized the expression of seven stress-response genes (proX, opuAC, mnhE, nhaC, trkH, ducA, and pimT). This work represents the first systematic validation of RGs under polyextreme stress conditions, providing essential guidelines for future gene expression studies in extreme environments and aiding research on microbial adaptation mechanisms in halophilic, alkaliphilic, and thermophilic microorganisms. Full article

Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions. Consistently, PIMT levels were reduced in the pancreatic islets isolated from high fat diet (HFD)-fed mice. The RNA sequencing analysis of PIMT knockdown beta cells identified that the expression of key genes involved in insulin secretory pathway, Ins1 (insulin 1), Ins2 (insulin 2), Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Kcnn1 (potassium calcium-activated channel subfamily N member 1), Rab3a (member RAS oncogene family), Gnas (GNAS complex locus), Syt13 (synaptotagmin 13), Pax6 (paired box 6), Klf11 (Kruppel-Like Factor 11), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1) was attenuated due to PIMT depletion. PIMT ablation in the pancreatic beta cells and in the rat pancreatic islets led to decreased protein levels of PDX1 and MafA, resulting in the reduction in glucose-stimulated insulin secretion (GSIS). The results from the immunoprecipitation and ChIP experiments revealed the interaction of PIMT with PDX1 and MafA, and its recruitment to the insulin promoter, respectively. Importantly, PIMT ablation in beta cells resulted in the nuclear translocation of insulin. Surprisingly, forced expression of PIMT in beta cells abrogated GSIS, while Ins1 and Ins2 transcript levels were subtly enhanced. On the other hand, the expression of genes, PRIP/Asc2/Ncoa6 (nuclear receptor coactivator 6), Pax6, Kcnj11, Syt13, Stxbp1 (syntaxin binding protein 1), and Snap25 (synaptosome associated protein 25) associated with insulin secretion, was significantly reduced, providing an explanation for the decreased GSIS upon PIMT overexpression. Our findings highlight the importance of PIMT in the regulation of insulin synthesis and secretion in beta cells. Full article

Seed ageing is an important issue for the long-time seed storage of tobacco. Seed priming has been popularly applied in tobacco production. In this study, the development of molecular marker genes encoding proteins L-isoaspartyl methyltransferase NtPIMT1 and 8-oxoG DNA glycosylase 1 NtOGG1 to detect the degree of seed ageing and the effect of seed priming is conducted in tobacco. Quantitative real-time PCR (qRT-PCR) analysis reveals that relatively higher mRNAs of NtPIMT1 and NtOGG1 are observed in the dry and early germinating seeds. The expressions of NtPIMT1 and NtOGG1 are negatively correlated with the degree of seed damage in non-pelleted and pelleted seeds after accelerated ageing treatments. The early best effects of gibberellin (GA₃) priming on speed and uniform germination are observed in 33 h primed seeds, and relatively lower expressions of NtPIMT1 and NtOGG1 are observed in priming seeds. NtPIMT1 and NtOGG1 genes have potential for use as molecular markers in detecting the seed ageing degree and priming effect of tobacco. Full article

The enzyme PIMT methylates abnormal aspartyl residues in proteins. U-87 MG cells are commonly used to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT isoform I possessed oncogenic features when overexpressed in U-87 MG and U-251 MG glioma cells. Higher levels of wild-type PIMT stimulated migration and invasion in both glioma cell lines. Conversely, PIMT silencing reduced these migratory abilities of both cell lines. These results indicate that PIMT could play a critical role in glioblastoma growth. Here, we investigated for the first time, molecular mechanisms involving PIMT in the regulation of epithelial to mesenchymal transition (EMT) upon TGF-β1 treatments. Gene array analyses indicated that EMT genes but not PIMT gene were regulated in U-87 MG cells treated with TGF-β1. Importantly, PIMT silencing by siRNA inhibited in vitro migration in U-87 MG cells induced by TGF-β1. In contrast, overexpressed wild-type PIMT and TGF-β1 had additive effects on cell migration. When PIMT was inhibited by siRNA, this prevented Slug induction by TGF-β1, while Snail stimulation by TGF-β1 was increased. Indeed, overexpression of wild-type PIMT led to the opposite effects on Slug and Snail expression dependent on TGF-β1. These data highlighted the importance of PIMT in the EMT response dependent on TGF-β1 in U-87 MG glioma cells by an antagonist regulation in the expression of transcription factors Slug and Snail, which are critical players in EMT. Full article

In this paper, we propose the first analytical procedure—using a screen-printed carbon electrode modified with carbon nanofibers (SPCE/CNFs)—for the detection and quantitative determination of an electroactive disubstituted fused triazinone, namely 4-Cl-PIMT, which is a promising anticancer drug candidate. The electrochemical performances of the sensor were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and square-wave adsorptive stripping voltammetry (SWAdSV). The presence of carbon nanofibers on the sensor surface caused a decrease in charge-transfer resistance and an increase in the active surface compared to the bare SPCE. Under the optimised experimental conditions, the proposed voltammetric procedure possesses a good linear response for the determination of 4-Cl-PIMT in the two linear ranges of 0.5–10 nM and 10–100 nM. The low limits of detection and quantification were calculated at 0.099 and 0.33 nM, respectively. In addition, the sensor displays high reproducibility and repeatability, as well as good selectivity. The selectivity was improved through the use of a flow system and a short accumulation time. The SWAdSV procedure with SPCE/CNFs was applied to determine 4-Cl-PIMT in human serum samples. The SWAdSV results were compared to those obtained by the ultra-high-performance liquid chromatography coupled with electrospray ionization/single-quadrupole mass spectrometry (UHPLC-ESI-MS) method. Full article

Seed storability is a main agronomically important trait to assure storage safety of grain and seeds in rice. Although many quantitative trait loci (QTLs) and associated genes for rice seed storability have been identified, the detailed genetic mechanisms of seed storability remain unclear in rice. In this study, a genome-wide association study (GWAS) was performed in 456 diverse rice core collections from the 3K rice genome. We discovered the new nine QTLs designated as qSS1-1, qSS1-2, qSS2-1, qSS3-1, qSS5-1, qSS5-2, qSS7-1, qSS8-1, and qSS11-1. According to the analysis of the new nine QTLs, our results could well explain the reason why seed storability of indica subspecies was superior to japonica subspecies in rice. Among them, qSS1-2 and qSS8-1 were potentially co-localized with a known associated qSS1/OsGH3-2 and OsPIMT1, respectively. Our results also suggest that pyramiding breeding of superior alleles of these associated genes will lead to new varieties with improved seed storability in the future. Full article

Human carbonic anhydrase IX (CAIX), a unique member of the α carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function. Full article

Multiple co-localized paralogs of genes in Polypedilum vanderplanki’s genome have strong transcriptional response to dehydration and considered to be a part of adaptation machinery at the larvae stage. One group of such genes represented by L-isoaspartate O-methyltransferases (PIMT). In order to highlight specific role of PIMT paralogization in desiccation tolerance of the larvae we annotated and compared S-adenosylmethionine (SAM) dependent methyltransferases of four insect species. From another side we applied co-expression analysis in desiccation/rehydration time course and showed that PIMT coding genes could be separated into five clusters by expression profile. We found that among Polypedilum vanderplanki’s PIMTs only PIMT1 and PIMT2 have enzymatic activity in normal physiological conditions. From in silico analysis of the protein structures we found two highly variable regions outside of the active center, but also amino acid substitutions which may affect SAM stabilization. Overall, in this study we demonstrated features of Polypedilum vanderplanki’s PIMT coding paralogs related to different roles in desiccation tolerance of the larvae. Our results also suggest a role of different SAM-methyltransferases in the adaptation, including GSMT, JHAMT, and candidates from other classes, which could be considered in future studies. Full article

PIMT/NCOA6IP, a transcriptional coactivator PRIP/NCOA6 binding protein, enhances nuclear receptor transcriptional activity. Germline disruption of PIMT results in early embryonic lethality due to impairment of development around blastocyst and uterine implantation stages. We now generated mice with Cre-mediated cardiac-specific deletion of PIMT (csPIMT^−/−) in adult mice. These mice manifest enlargement of heart, with nearly 100% mortality by 7.5 months of age due to dilated cardiomyopathy. Significant reductions in the expression of genes (i) pertaining to mitochondrial respiratory chain complexes I to IV; (ii) calcium cycling cardiac muscle contraction (Atp2a1, Atp2a2, Ryr2); and (iii) nuclear receptor PPAR- regulated genes involved in glucose and fatty acid energy metabolism were found in csPIMT^−/− mouse heart. Elevated levels of Nppa and Nppb mRNAs were noted in csPIMT^−/− heart indicative of myocardial damage. These hearts revealed increased reparative fibrosis associated with enhanced expression of Tgfβ2 and Ctgf. Furthermore, cardiac-specific deletion of PIMT in adult mice, using tamoxifen-inducible Cre-approach (TmcsPIMT^−/−), results in the development of cardiomyopathy. Thus, cumulative evidence suggests that PIMT functions in cardiac energy metabolism by interacting with nuclear receptor coactivators and this property could be useful in the management of heart failure. Full article