Journal of biochemistry最新文献

Dementia with Lewy bodies (DLB) is the second most common neurodegenerative dementia after Alzheimer's disease (AD). Early differentiation of these disorders is crucial for managing core symptoms; however, existing biomarkers remain insufficient. DLB shares motor and cognitive symptoms with Parkinson's disease (PD), and both are classified as synucleinopathies due to abnormal α-synuclein aggregation. Although α-synuclein is predominantly expressed in the central nervous system, it is also abundant in erythrocytes. Recent studies suggest a potential link between erythrocyte-derived α-synuclein and synucleinopathy pathology. Additionally, we previously reported that both erythrocytes and circulating medium and large extracellular vesicles (m/lEVs) in plasma from healthy subjects contain full-length and C-terminally truncated α-synuclein. In this study, we found that erythrocyte α-synuclein levels were significantly lower in DLB compared to AD, PD and healthy controls. Furthermore, α-synuclein levels in circulating m/lEVs were elevated in patients with neurodegenerative diseases. These findings provide new insights into the role of peripheral α-synuclein and suggest its potential utility as a diagnostic marker for DLB. While further validation is needed, erythrocyte-derived α-synuclein may complement nuclear medicine assessments in distinguishing DLB from other neurodegenerative disorders.

ATP-dependent (S)-NAD(P)H-hydrate dehydratase (NAXD) is a crucial enzyme in the nicotinamide adenine dinucleotide repair system that regenerates NAD(P)H, an essential electron donor in metabolic redox reactions. NAD+-related metabolic pathways connect cellular metabolism and the expression of genes responsible for adipogenesis; however, the biological significance of the NAXD-mediated repair pathway remains unclear. Herein, we showed that NAXD is essential for normal adipocyte differentiation of 3T3-L1 murine preadipocytes. Silencing of the Naxd gene attenuated differentiation-induced lipid accumulation with excessive accumulation of hydrated NADH (NADHX) without altering NAD+ levels. FK866, a specific inhibitor of NAMPT, further reduced lipid accumulation even in Naxd-silenced cells with substantial decrease in NAD+. Supplementation with nicotinamide mononucleotide, a precursor of NAD+, restored NAD+ levels comparably in Naxd- and LacZ-silenced cells treated with FK866, but failed to recover adipocyte differentiation of Naxd-silenced cells to the level of LacZ-silenced cells. In contrast, exposure of wild-type 3T3-L1 cells to NADHX recapitulated the Naxd deficiency-elicited inhibitory effects on adipocyte differentiation with reduced expression of master transcriptional regulators of adipogenesis, peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α. These results suggest that NAXD supports normal adipogenesis, in part, by inhibiting excessive accumulation of NADHX.

The reverse transcription (RT) of RNA to cDNA is a key step for the quantification of nucleic acid molecules in numerous basic research and medical diagnosis. Although multiple sources of errors have been considered, little is known about the impact of RNA modifications on the validity of genes of interest for quantitative RT-PCR. Here, we evaluated the influence of RNA modifications of N1-methyladenosine (m1A) on the validity of the RT step by quantifying two RNAs with commercial reverse transcriptase and RNA sample from HEK-293 T cells or in vitro transcription. Our findings prove that RNA modification of m1A is a source of RT variability as it acts as an arrest signal of RT at its position, in turn affecting the corresponding RNA quantification.

The uterine endometrium consists of luminal epithelium, glandular epithelium and stromal cells, with uterine glands playing a pivotal role in pregnancy success among mammals. Uterine glands secrete essential factors that regulate embryo development and implantation; however, their cellular biology remains poorly understood. This study presents a refined method for isolating three distinct endometrial cell types with high purity, with a specific emphasis on glandular epithelial (GE) cells. The method combines mechanical dissociation, enzymatic digestion and immunomagnetic separation. The isolated GE cells were maintained in culture and exhibited proliferation in response to steroid hormones. Furthermore, oestrogen responsiveness was abrogated by Estrogen Receptor 1 (Esr1) knockdown mediated by siRNA. Here, we present an efficient and reproducible method for isolating uterine GE cells with high purity, enabling their in vitro maintenance, hormone responsiveness assessment and functional gene knockdown. These findings establish a robust platform for advancing our understanding of uterine gland biology, facilitating detailed investigations into molecular mechanisms underlying glandular function and their critical roles in establishing pregnancy success. Future research could explore the contribution of these isolated cells to endometrial receptivity and embryo implantation.

Small GTPases act as molecular switches regulating various cellular processes by cycling between the GDP- and GTP-bound states. Several methods, including radioisotope-based nucleotide exchange assays, effector-binding pull-down assays and fluorescence-based biosensor methods, have been developed to assess the activation of small GTPases. In vitro techniques mainly provide quantitative insights, whereas live-cell imaging approaches facilitate the real-time monitoring of the activation dynamics of small GTPases. Recent advances, such as the development of fluorescence resonance energy transfer-based probes and membrane-localization sensors, have improved the spatial and temporal resolution of small GTPase activation dynamics. Specifically, the small GTPase activity analysing system using a split fluorescent protein to detect membrane recruitment upon activation provides a novel approach to study small GTPases in living cells. This review comprehensively discusses various conventional and emerging small GTPase activation analysis techniques, highlighting their advantages and disadvantages in studying small GTPase activation dynamics under different cellular conditions.

Mitochondria are believed to have originated from the endosymbiosis of bacteria and they still contain their own genome, which is called mitochondrial DNA (mtDNA). Under fluorescence microscopy of cultured mammalian cells, mtDNA is observed as numerous tiny dot-like structures called mitochondrial nucleoids. In live-imaging, the morphology and distribution of nucleoids are changed dynamically, but the molecular details remain poorly understood. In this study, we constructed a custom siRNA library targeting 1,164 human mitochondria-related genes, and from live-imaging-based screening of HeLa cells, we identified that mitochondrial calcium uniporter (MCU), a pore-forming subunit of the mitochondrial Ca2+ channel, is involved in nucleoid morphology. We found that suppression of MCU by RNAi induced the formation of highly enlarged nucleoids as well as respiratory dysfunction and that the re-introduction of MCU or treatment with Ca2+ ionophore recovered the enlarged nucleoid morphology. These results suggest that mitochondrial Ca2+ uptake via MCU is associated with nucleoid morphology. The constructed siRNA library might be widely applied to analyze the roles of mitochondrial proteins in various cellular events, making it useful to understand the multifaceted functions of mitochondria in human cells.

Heterochromatin plays an important role in eukaryotic cellular functions, including gene silencing, higher-order chromatin structure, genome stability and so on. Heterochromatin protein 1 (HP1), a key component of heterochromatin, is conserved from fission yeast to mammals. HP1 binds to histone H3K9me, a hallmark of heterochromatin, through its N-terminal chromodomain (CD) and self-dimerizes and recruits other chromatin proteins through its C-terminal chromo shadow domain (CSD), acting as an epigenetic reader. Between the CD and CSD is an unstructured, less conserved hinge region, which has been implicated in nucleic acid binding. The molecular dissection of the fission yeast HP1 orthologue, Chp2, recently reported in this journal, elucidated the cooperative DNA binding of the hinge and N-terminus of the CSD, which contributes to the stable association with heterochromatin and gene silencing. In this commentary, we focus on the mechanisms involving the HP1 hinge region, which is more than a simple linker.

Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipids in plants. Previously, we found phospholipase D (PLD) activity that hydrolyzes GIPC to phytoceramide 1-phosphate (PCerP) in plants and revealed that GIPC-PLD activity is carried out by an enzyme encoded by non-specific phospholipase C3 (NPC3) gene. In this study, we established a method for isolation and quantification of inositol glycan (InoGly), a counterpart of PCerP produced from GIPC, using TLC imaging. We confirmed that Arabidopsis thaliana NPC3 protein and partially purified GIPC-PLD from cabbage produced InoGly in a similar amount to that of PCerP from purified GIPC. We applied our method to determination of InoGly present in plant tissues and found that it was present at 40-80 nmol/g (wet weight) in cabbage leaves, radish root and broccoli stem and increased to 80-120 nmol/g after homogenization of the tissues. Similar increases in PCerP and decreases in GIPC were observed after homogenization, indicating that InoGly and PCerP were produced from GIPC by GIPC-PLD activity in response to homogenization. We believe our method, which does not require a complicated process or large device, will contribute to a better understanding of GIPC metabolism and signalling in plants.

Two genes, AML-I and AML-II, have been reported to exhibit increased expression during the development of the coral Acropora millepora. They show amino acid sequence homology with CEL-III, a haemolytic lectin found in the sea cucumber Cucumaria echinata. CEL-III binds to carbohydrate chains on the surface of erythrocytes, forming heptameric pores in their membranes. To clarify the role of these proteins in coral, we identified and elucidated their functions. The carbohydrate-binding domains of them showed similar carbohydrate-binding specificity as that of CEL-III. AML-I showed haemagglutinating activity in erythrocytes, whereas AML-II can only be prepared as an aggregate and its function could not yet be determined. AML-IΔC and AML-IIΔC mutants were generated through deletion of the C-terminal extended amino acid residues of them relative to CEL-III. AML-IΔC showed haemolytic activity towards erythrocytes, whereas AML-IIΔC showed no activity. A tobacco etch virus (TEV) protease recognition site was inserted into the C-terminus of AML-I to regulate these activities. The haemagglutinating activity of AML-I was converted into haemolytic activity after TEV protease treatment. As a result, TEV protease could control the haemolytic and haemagglutinating activity of the lectin, which could be useful as an anticancer or antiviral drug because of its cytotoxic activity.

Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a novel oncogene/oncoprotein that downregulates the p53 tumour suppressor protein through several mechanisms. One important mechanism involves binding of GRWD1 to RPL11, which competitively inhibits the RPL11-MDM2 interaction and releases RPL11-mediated suppression of MDM2 ubiquitin ligase activity towards p53. Here, we mined the TCGA (The Cancer Genome Atlas) database to gain in-depth insight into the clinical relevance of GRWD1. We found that high expression of GRWD1 is associated with a poor prognosis for lower grade glioma (LGG) of the brain, skin cutaneous melanoma (SKCM), and kidney renal clear cell carcinoma (KIRC) carrying wild-type p53. Further investigations revealed that copy number alterations in the GRWD1 gene are one determinant of GRWD1 expression level. By contrast, even in patients with a diploid GRWD1 gene, high GRWD1 expression was associated with a poor prognosis for LGG, SKCM, and KIRC carrying wild-type p53. Additional studies suggest that some transcriptional factors may be involved in regulation of GRWD1 in cancers with a diploid GRWD1 gene. Taken together, the data presented herein suggest that high expression of GRWD1 may contribute to malignant behaviour, and predict a clinically unfavourable prognosis for LGG, SKCM, and KIRC carrying wild-type p53.