Morphological Changes In Nucleus Research Articles

Cell redistribution of G quadruplex-structured DNA is associated with morphological changes of nuclei and nucleoli in neurons during tau pathology progression.

While the double helical structure has long been its iconic representation, DNA is structurally dynamic and can adopt alternative secondary configurations. Specifically, guanine-rich DNA sequences can fold in guanine quadruplexes (G4) structures. These G4 play pivotal roles as regulators of gene expression and genomic stability, and influence protein homeostasis. Despite their significance, the association of G4 with neurodegenerative diseases such as Alzheimer's disease (AD) has been underappreciated. Recent findings have identified DNA sequences predicted to form G4 in sarkosyl-insoluble aggregates from AD brains, questioning the involvement of G4-structured DNA (G4 DNA) in the pathology. Using immunofluorescence coupled to confocal microscopy analysis we investigated the impact of tau pathology, a hallmark of tauopathies including AD, on the distribution of G4 DNA in murine neurons and its relevance to AD brains. In healthy neurons, G4 DNA is detected in nuclei with a notable presence in nucleoli. However, in a transgenic mouse model of tau pathology (THY-Tau22), early stages of the disease exhibit an impairment in the nuclear distribution of G4 DNA. In addition, G4 DNA accumulates in the cytoplasm of neurons exhibiting oligomerized tau and oxidative DNA damage. This altered distribution persists in the later stage of the pathology when larger tau aggregates are present. Still cytoplasmic deposition of G4 DNA does not appear to be a critical factor in the tau aggregation process. Similar patterns are observed in neurons from the AD cortex. Furthermore, the disturbance in G4 DNA distribution is associated with various changes in the size of neuronal nuclei and nucleoli, indicative of responses to stress and the activation of pro-survival mechanisms. Our results shed light on a significant impact of tau pathology on the dynamics of G4 DNA and on nuclear and nucleolar mechanobiology in neurons. These findings reveal new dimensions in the etiopathogenesis of tauopathies.

Synthesis of sinomenine derivatives with potential anti-leukemia activity

In recent years, with sinomenine hydrochloride as the main ingredient, Qingfengteng had been formulated as various dosage forms for clinical treatment. Subsequent findings confirmed a variety of biological roles for sinomenine. Here, 15 H2S-donating sinomenine derivatives were synthesized. Target hybrids a11 displayed substantial cytotoxic effects on cancer cell lines, particularly against K562 cells, with an IC50 value of 1.36 μM. In-depth studies demonstrated that a11 arrested cell cycle at G1 phase, induced apoptosis via both morphological changes in nucleus and membrane potential collapse in mitochondria. These results indicated a11 exerted an antiproliferative effect through apoptosis induction via mitochondrial pathway.

Ni/Mn-Complex-Tethered Tetranuclear Polyoxovanadates: Crystal Structure and Inhibitory Activity on Human Hepatocellular Carcinoma (HepG-2).

Polyoxometalates (POMs) exhibit unique structural characteristics and excellent physical and chemical properties, which have attracted significant attention from scholars in the fields of anticancer research and chemotherapy. Herein, we successfully synthesized and structurally characterized two novel polyoxovanadates (POVs), denoted as POVs-1 and POVs-2, where [M(1-vIM)4]2[VV4O12]·H2O (M: NiII and MnII, 1-vinylimidazole abbreviated as 1-vIM) serve as ligands. The two POVs are isomeric and consist of fundamental structural units, each comprising one [V4O12]4- cluster, two [M(1-vIM)4]2+ cations, and one water molecule. Subsequently, we evaluated the cell viability of human hepatocellular carcinoma (HepG-2) cells treated with the synthesized POVs using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay. And the changes in cell nucleus morphology, mitochondrial membrane potential (Δψm), and reactive oxygen species levels in HepG-2 exposed to POVs were monitored using specific fluorescent staining techniques. Both hybrid POVs showed potent inhibitory activities, induing apoptosis in HepG-2 cells along with significant mitochondria dysfunction and a burst of reactive oxygen species. Notably, the inhibitory effects of POVs-2 were more pronounced than those of POVs-1, which is primarily attributed to the different transition metal ions present. These findings underscore the intricate relationship between the metal components, structural characteristics, and the observed antitumor activities in HepG-2 cells.

Nucleus Mechanosensing in Cardiomyocytes.

Cardiac muscle contraction is distinct from the contraction of other muscle types. The heart continuously undergoes contraction-relaxation cycles throughout an animal's lifespan. It must respond to constantly varying physical and energetic burdens over the short term on a beat-to-beat basis and relies on different mechanisms over the long term. Muscle contractility is based on actin and myosin interactions that are regulated by cytoplasmic calcium ions. Genetic variants of sarcomeric proteins can lead to the pathophysiological development of cardiac dysfunction. The sarcomere is physically connected to other cytoskeletal components. Actin filaments, microtubules and desmin proteins are responsible for these interactions. Therefore, mechanical as well as biochemical signals from sarcomeric contractions are transmitted to and sensed by other parts of the cardiomyocyte, particularly the nucleus which can respond to these stimuli. Proteins anchored to the nuclear envelope display a broad response which remodels the structure of the nucleus. In this review, we examine the central aspects of mechanotransduction in the cardiomyocyte where the transmission of mechanical signals to the nucleus can result in changes in gene expression and nucleus morphology. The correlation of nucleus sensing and dysfunction of sarcomeric proteins may assist the understanding of a wide range of functional responses in the progress of cardiomyopathic diseases.

Rapid Screening of Urinary Tract Infection Using Microfluidic Inertial-Impedance Cytometry.

Urinary tract infection (UTI) diagnosis based on urine culture for bacteriuria analysis is time-consuming and often leads to wastage of hospital resources due to false-positive UTI cases. Direct cellular phenotyping (e.g., RBCs, neutrophils, epithelial cells) of urine samples remains a technical challenge as low cell concentrations, and urine characteristics (conductivities, pH, microbes) can affect the accuracy of cell measurements. In this work, we report a microfluidic inertial-impedance cytometry technique for label-free rapid (<5 min) neutrophil sorting and impedance profiling from urine directly. Based on size-based inertial focusing effects, neutrophils are isolated, concentrated, and resuspended in saline (buffer exchange) to improve consistency in impedance-based single-cell analysis. We first observed that both urine pH and the presence of bacteria can affect neutrophil high-frequency impedance measurements possibly due to changes in nucleus morphology as neutrophils undergo NETosis and phagocytosis, respectively. As a proof-of-concept for clinical testing, we report for the first time, rapid UTI testing based on multiparametric impedance profiling of putative neutrophils (electrical size, membrane properties, and distribution) in urine samples from non-UTI (n = 20) and UTI patients (n = 20). A significant increase in cell count was observed in UTI samples, and biophysical parameters were used to develop a UTI classifier with an area under the receiver operating characteristic curve of 0.84. Overall, the developed platform facilitates rapid culture-free urine screening which can be further developed to assess disease severity in UTI and other urologic diseases based on neutrophil electrical signatures.

Development of Novel Pyridine-Thiazole Hybrid Molecules as Potential Anticancer Agents.

Novel pyridine-thiazole hybrid molecules were synthesized and subjected to physico-chemical characterization and screening of their cytotoxic action towards a panel of cell lines derived from different types of tumors (carcinomas of colon, breast, and lung, glioblastoma and leukemia), and normal human keratinocytes, for comparison. High antiproliferative activity of the 3-(2-fluorophenyl)-1-[4-methyl-2-(pyridin-2-ylamino)-thiazol-5-yl]-propenone 3 and 4-(2-{1-(2-fluorophenyl)-3-[4-methyl-2-(pyridin-2-ylamino)-thiazol-5-yl]-3-oxopropylsulfanyl}-acetylamino)-benzoic acid ethyl ester 4 was revealed. The IC50 of the compound 3 in HL-60 cells of the acute human promyelocytic leukemia was 0.57 µM, while in the pseudo-normal human cell lines, the IC50 of this compound was >50 µM, which suggests that the compounds 3 and 4 might be perspective anticancer agents. The detected selectivity of the derivatives 3 and 4 for cancer cell lines inspired us to study the mechanisms of their cytotoxic action. It was shown that preincubation of tumor cells with Fluzaparib (inhibitor of PARP1) reduced the cytotoxic activity of the derivatives 3 and 4 by more than twice. The ability of these compounds to affect DNA nativity and cause changes in nucleus morphology allows for the suggestion that the mechanism of action of the novel pyridine-thiazole derivatives might be related to inducing the genetic instability in tumor cells.

Ethyl Acetate Fraction of Hedyotis diffusa Willd Induces Apoptosis via JNK/Nur77 Pathway in Hepatocellular Carcinoma Cells.

Background Hepatocellular carcinoma (HCC) is characterized by poor diagnosis and high mortality. Novel and efficient therapeutic agents are urgently needed for the treatment. Hedyotis diffusa Willd (HDW) is used to treat cancers, especially HCC in China. Purpose The study aimed to identify the main anti-HCC extract in HDW and to explore the mechanism of the active extract. Materials and Methods The high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was used for the simultaneous determination of main compounds in the ethyl acetate fraction of HDW (EHDW). The toxicity test of different HDW fractions was carried out on larvae at 2 day-post-fertilization (dpf) for 72 h. The in vivo anti-HCC effect of different HDW fractions was evaluated on a zebrafish tumor model by immersion administration. The antiproliferative effect of HDW fractions was determined with MTT assay, as well as hematoxylin and eosin (HE) staining assay. Hoechst 33258 staining was used to observe changes in nucleus morphology. Flow cytometry analysis was used to investigate apoptosis induction. Western blot analysis was used to examine apoptosis-related proteins, and key proteins in JNK/Nur77 signaling pathway. SP600125 was served to validate the apoptotic mechanism. Results EHDW showed the strongest tumor cell growth inhibitory effect on zebrafish tumor model. Further study revealed that EHDW induced apoptosis in zebrafish tumor model and in cultured Hep3B cells. Meanwhile, it has been shown that the levels of BCL2-associated X (Bax), cytochrome c (cyto c), cleaved-caspase 3, and poly-ADP-ribose polymerase (PARP) cells were upregulated. In contrast, the level of antiapoptotic B cell lymphoma-2 (Bcl-2) was downregulated in Hep3B cells. Additionally, EHDW activated JNK/Nur77 pathway by increasing the levels of p-JNK(Thr183/Tyr185) and p-Nur77(Ser351). Further study showed that blockage of JNK by SP600125 reversed EHDW-induced JNK/Nur77 pathway and the downstream apoptotic proteins. Conclusion In conclusion, EHDW exerted the anti-HCC effect, which may be attributed to the activation of JNK/Nur77 pathway. This study supported the rationale of HDW as an HCC therapeutic agent.

Ricinus communis Butanol Fraction Inhibits MCF-7 Breast Cancer Cell Migration, Adhesion, and Invasiveness.

In this study, the potential of an n-butanol fraction from Ricinus communis to prevent metastasis in MCF-7 breast cancer cells was investigated. The effect of the fraction on BUD-8 and MCF-7 cell viability was assessed using the MTT assay. Apoptotic cell death was analyzed by Hoechst staining assay. The antimetastatic effect of the fraction on MCF-7 cell was evaluated using the wound healing, adhesion and Boyden chamber invasion assays. Gelatin-zymography was used to assess the effect of the fraction on MMP-2 and MMP-9 activity. The expression profile of proteins implicated in metastasis and angiogenesis was determined using the human angiogenesis antibody array kit, following treatment with the fraction. BUD-8 cell viability was significantly reduced at concentrations between 300 and 500 µg/ml of the extract. In contrast, a significant reduction in cell viability was seen in MCF-7 cells treated with 400 to 500 µg/ml of the fraction. At sub-lethal concentrations (100 and 200 µg/ml) of the fraction, no nuclei morphological changes associated with apoptotic cell death were observed in MCF-7 cells. In addition, the fraction showed to have an inhibitory effect on MCF-7 cell migration, adhesion, invasiveness, and MMP-2 activity. Moreover, the fraction was seen to modulate the expression of several proteins, such as MMP-9, uPA, VEGF, and TGF-β1, playing a role in the metastasis process. This study demonstrates that the n-butanol fraction of R. communis can inhibit major steps of the metastatic cascade and modulate metastasis regulatory proteins. Thus, the fraction can be considered a potential source of antimetastatic agents that could be useful in the treatment of malignant cancers.

Seasonal changes of perineuronal nets and song learning in adult canaries (Serinus canaria)

Songbirds learn their song during a sensitive period of development associated with enhanced neural plasticity. In addition, in open-ended learners such as canaries, a sensitive period for sensorimotor vocal learning reopens each year in the fall and leads to song modifications between successive breeding seasons. The variability observed in song production across seasons in adult canaries correlates with seasonal fluctuations of testosterone concentrations and with morphological changes in nuclei of the song control system (SCS). The sensitive periods for song learning during ontogeny and then again in adulthood could be controlled by the development of perineuronal nets (PNN) around parvalbumin-expressing interneurones (PV) which limits learning-induced neuroplasticity. However, this relationship has never been investigated in the context of adult vocal learning in adult songbirds. Here we explored PNN and PV expression in the SCS of adult male Fife Fancy canaries in relation to the seasonal variations of their singing behaviour. We found a clear pattern of seasonal variation in testosterone concentrations and song production. Furthermore, PNN expression was significantly higher in two specific song control nuclei, the robust nucleus of the arcopallium (RA) and the Area X of the basal ganglia, during the breeding season and during the later stages of sensorimotor song development compared to birds in an earlier stage of sensorimotor development during the fall. These data provide the first evidence that changes in PNN expression could represent a mechanism regulating the closing-reopening of sensitive periods for vocal learning across seasons in adult songbirds.

Morphological Characteristics of Neuronal Death After Experimental Subarachnoid Hemorrhage in Mice Using Double Immunoenzymatic Technique.

Subarachnoid hemorrhage (SAH) is a devastating disease. Neuronal death is an important pathophysiology in the acute phase of SAH, but the histopathological features of dying neurons have been poorly studied. Using several staining methods including terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and microtubule-associated protein 2 (MAP-2) double immunolabeling, we investigated the morphological changes of nucleus and cytoskeleton in neurons and sought susceptible areas to neuronal death in filament perforation SAH mice under light microscope. TUNEL and MAP-2 double immunolabeling clearly showed morphological features of shrunken cytoplasm and sometimes curl-like fibers in dying neurons, besides nuclear abnormalities. More dying neurons were detected in the moderate SAH group than in the mild SAH group, and the temporal base cortex was the most susceptible area to neuronal death with deoxyribonucleic acid (DNA) damage among the cerebral cortices and hippocampus at 24 hr after SAH (p<0.01, ANOVA). Lesser hippocampal neuronal death was observed at 24 hr, but neuronal death was significantly increased in the CA1 region at 7 days after SAH (p<0.05, unpaired t-test). Using TUNEL and MAP-2 double immunolabeling, morphological features of not only the nucleus but also the cytoplasm in post-SAH neuronal death with DNA damage can be observed in detail under light microscope.

Synthesis and anticancer evaluation of benzo-N-heterocycles transition metal complexes against esophageal cancer cell lines

Three novel transition metal complexes, Cu(p-2-bmq)Cl2 (1), Zn(p-2-bmq)Cl2 (2) and [Co(p-2-bmq)Cl2]2 (3) (where p-2-bmq = 2-((1-(pyridin-2-yl)-1H-benzoimidazol-2-yl)methyl) quinolone, have been synthesized. The complexes were detected for their cytotoxicity in vitro against four human esophageal cancer cell lines (SMMC7721, BGC823, HCT116 and HT29) by MTT assay. The results showed that they all have anti-tumor cell proliferation activity. E specially, complex 1 exhibited significant cytotoxicity with IC50 value of 15.89 μM against SMMC7721 cells for 72 h. The morphological changes of nuclei by fluorescence staining methods proved that complex 1 could induce intracellular DNA damage. The flow cytometry analysis revealed that the treatment of SMMC7721 cells with complex 1 induced intracellular ROS increased, mitochondrial potential collapse, G2/M-phase arrest, and even apoptosis. These studies should highly valuable for the development of transition metal-based compounds to the potential anticancer medicinal applications.

Investigating the Effects of Morphine on Survival and Sensitivity to Cisplatin in Ovarian Cancer Cells

Morphine as an analgesic drug is used frequently in cancer patients. Contradictory results have been achieved from previous studies related to morphine effects in different concentrations. In current study, we examined the effect of clinical concentrations of morphine on A2780Cp cell line related to ovarian cancer. Moreover, its effect on the cytotoxicity of cisplatin was investigated. A2780CP cells were cultured in RPMI1640 medium and treated with clinical doses of morphine alone or in combination with cisplatin. The rate of cell proliferation was measured using MTT assay, morphological changes of nuclei were revealed by 4′,6-diamidino-2-phenylindole (DAPI) staining, and expression of B-cell lymphoma 2 (Bcl-2) was measured using flowcytometry. MTT assay results showed clinical concentration of morphine had no effect on viability of A2780CP cells and toxicity of cisplatin. DAPI staining revealed no chromatin condensation in presence of morphine, and flowcytometry analysis showed that the expression of Bcl-2 in treated cells did not differ from control cells. In accordance with findings in other kinds of cancer, our results demonstrated that morphine did not interact with the function of cispatin in ovarian cancer. This finding can be considered in clinical applications of morphine.

Cytotoxicity of the natural herbicidal chemical, berberine, on Nicotiana tabacum Bright yellow-2 cells

Berberine is a naturally occurring plant secondary metabolite with allelopathic and cytotoxic properties. We investigated the cytotoxic effects of berberine against tobacco Bright Yellow-2 (BY-2) cells. We found that berberine inhibited tobacco BY-2 cell growth in a concentration-dependent manner and the potency of berberine was comparable to the traditional herbicide glyphosate. Meanwhile, the in vivo test revealed that the herbicidal activity of berberine was also comparable to that of glyphosate. Further mechanism studies for the cytotoxicity demonstrated that berberine at concentrations of 40 μg/mL and 80 μg/mL induced cell death by causing mitochondrial membrane depolarization, irregular nuclei and chromatin condensation but not leading to significant apoptosis. Ultra-structure analysis through transmission electron microscopy (TEM) indicated that treatments with 40 μg/mL berberine for 2 d or 80 μg/mL berberine for 1 d induced cell damage, including nuclei morphological changes, cytoplasm and mitochondria degradation and cell wall fibrosis. Treatment at higher concentration of 80 μg/mL for longer period of 2 d, induced plasmolysis and severe damage to basic cell structure, which are indicative of explosive necrosis. Our results suggest that berberine was cytotoxic to tobacco BY-2 cells by inducing necrosis but not apoptosis. Cell wall, mitochondria, nuclei and chromatin were damaged and may be possible primary targets. Therefore, the herbicidal activity of berberine appears to be a complex process associated with multiple mechanisms of action.

Activation of Nucleases, PCD, and Mobilization of Reserves in the Araucaria angustifolia Megagametophyte During Germination.

The megagametophyte of mature seeds of Araucaria angustifolia consists of cells with thin walls, one or more nuclei, a central vacuole storing proteins, and a cytoplasm rich in amyloplasts, mitochondria and lipid bodies. In this study, we describe the process of mobilization of reserves and analyzed the dismantling of the tissue during germination, using a range of well-established markers of programmed cell death (PCD), including: morphological changes in nuclei and amyloplasts, DNA degradation, and changes in nuclease profiles. TUNEL reaction and DNA electrophoresis demonstrate that DNA fragmentation in nuclei occurs at early stages of germination, which correlates with induction of specific nucleases. The results of the present study add knowledge on the dismantling of the megagametophyte of genus Araucaria, a storage tissue that stores starch as the main reserve substance, as well as on the PCD pathway, by revealing new insights into the role of nucleases and the expression patterns of putative nuclease genes during germination.

Temporal Changes in Nucleus Morphology, Lamin A/C and Histone Methylation During Nanotopography-Induced Neuronal Differentiation of Stem Cells.

Stem cell differentiation can be regulated by biophysical cues such as nanotopography. It involves sensing and integration of these biophysical cues into their transcriptome with a mechanism that is yet to be discovered. In addition to the cytoskeletal and focal adhesion remodeling, nanotopography has also been shown to modulate nucleus morphology. Here, we studied the effect of nanotopography on the temporal changes in nuclei of human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs). Using a high throughput Multi-architecture (MARC) chip analysis, the circularity of the stem cell nuclei changed significantly on different patterns. Human ESCs and MSCs showed different temporal changes in nucleus morphology, lamin A/C expression and histone methylation during topography-induced neuronal differentiation. In hESCs, the expression of nuclear matrix protein, lamin A/C during neuronal differentiation of hESCs on PDMS samples were weakly detected in the first 7 days of differentiation. The histone 3 trimethylation on Lysine 9 (H3K9me3) decreased after differentiation initiated and showed temporal changes in their expression and organization during neuronal differentiation. In hMSCs, the expression of lamin A/C was significantly increased after the first 24 h of cell culture. The quantitative analysis of histone methylation also showed a significant increase in hMSCs histone methylation on 250 nm anisotropic nanogratings within the first 24 h of seeding. This reiterates the importance of cell-substrate sensing within the first 24 h for adult stem cells. The lamin A/C expression and histone methylation shows a correlation of epigenetic changes in early events of differentiation, giving an insight on how extracellular nanotopographical cues are transduced into nuclear biochemical signals. Collectively, these results provide more understanding into the nuclear regulation of the mechanotransduction of nanotopographical cues in stem cell differentiation.

Antitumor activity of chitosan from mayfly with comparison to commercially available low, medium and high molecular weight chitosans.

Insects' cuticles have a potential to be evaluated as a chitin source. Especially adults of aquatic insects like mayflies (order Ephemeroptera) swarm in enormous numbers in artificially lit areas while mating in spring and then die by leaving huge amounts of dead insects' bodies. Here in this study, mayfly corpses were harvested and used for production of low MW chitosan. Dried mayfly bodies had 10.21% chitin content; mayfly chitin was converted into chitosan with efficiency rate of 78.43% (deacetylation degree, 84.3%; MW, 3.69kDa). Cytotoxicity and anti-proliferative activity of mayfly and commercially available shrimp chitosans (low, medium, and high MW) were determined on L929 fibroblast and three different cancer types including HeLa, A549, and WiDr. Apoptosis and necrosis stimulating potential of mayfly and commercial chitosans were also evaluated on A549 and WiDr cells using acridine orange and propidium iodide dual staining to observe morphological changes in nuclei and thus to reveal the predominant cell death mechanism. The effects of chitosans have varied depending on cell types, concentration, and chitosan derivatives. Mayfly and low MW chitosans had a cytotoxic effect at a concentration of 500μgmL-1 on non-cancer cells. At concentrations below this value (250μgmL-1), mayfly and commercial chitosans except high MW one exhibited strong inhibitory activity on cancer cells especially A549 and WiDr cells. Mayfly chitosan induced early and late apoptosis in A549 cells, but late apoptosis and necrosis in WiDr cells. This study suggests that dead bodies of mayflies can be used for production of low MW chitosan with anti-proliferative activity.

Naringenin alleviates high D-glucose-induced injuries through upregulation of microRNA-30d-5p level in human AC16 cardiac cells

As a common complication of diabetes mellitus (DM), diabetic cardiomyopathy (DCM) is considered to be one of the major causes of mortality and morbidity. The therapeutic effects of naringenin have been verified in the treatment of various human diseases. However, the application of naringenin in the treatment of DCM still has not been reported. In this study, human AC16 cardiac cells were treated with normal d-glucose and high d-glucose (HG). After transfection with miR-30d-5p inhibitor, Cell Counting Kit-8 (CCK-8) method was used to measure cell viability. Hoechst 33258 staining was performed to observe the morphological changes of nucleus. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the activity of caspase-3. Cell apoptosis was detected by Annexin V-FITC/propidium iodide (PI) staining. Levels of light chain 3 (LC3) including LC3-I and LC3-II as well as nucleoporin p62 (P62) were detected by Western blot. We found that Naringenin treatment increased the reduced cell variability caused by HG treatment. Naringenin also increased expression level of miR-30d-5p in human AC16 cardiac cells after HG treatment. Treatment with miR-30d-5p inhibitor reduced the effect of miR-30d-5p in increasing cell variability and reducing cell apoptosis. Naringenin treatment reduced the increased levels of LC-I, LC-II and P62, but miR-30d-5p inhibitor reduced those changes. Therefore we concluded that naringenin could alleviate HG-induced injuries through the upregulation of microRNA-30d-5p level in human AC16 cardiac cells.

Protective effects of Cervus nippon Temminck velvet antler polypeptides against MPP+‑induced cytotoxicity in SH‑SY5Y neuroblastoma cells.

The aim of the present study was to examine the protective effects and mechanism of sika deer (Cervus nippon Temminck) velvet antler polypeptides (VAPs) against MPP+ exposure in the SH-SY5Y human neuroblastoma cell line. MPP+ cytotoxicity and the protective effects of VAPs on the SH-SY5Y cells were determined using an MTT assay. Cell apoptosis and mitochondrial membrane potential were detected using Hoechst 33342 and Rhodamine123 staining, respectively. Endoplasmic reticulum (ER) stress-related reactive oxygen species (ROS) production in the SH-SY5Y cells was detected using 2′,7′-dichlorodihydrofluorescein diacetate fluorescent probes. The expression levels of proteins, including caspase-12, glucose regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP) and phosphorylated c-Jun N-terminal kinase (p-JNK) were detected using western blot analysis. The results showed that the half inhibitory concentration of MPP+ at 72 h was 120.9 µmol/l, and that 62.5, 125, and 250 µg/ml concentrations of VAPs protected the SH-SY5Y cells under MPP+ exposure. When exposed to 120.9 µmol/l MPP+, changes in cell nucleus morphology, mitochondrial membrane potential and intracellular ROS were observed. VAPs at concentrations of 62.5, 125, 250 µg/ml reduced this damage. Western blot analysis showed that protein expression levels of caspase-12, GRP78 and p-JNK were upregulated in the SH-SY5Y cells exposed to 120.9 µmol/l MPP+ for 72 h. In addition, 62.5, 125, and 250 µg/ml VAPs downregulated the expression levels of caspase-12 and p-JNK in a concentration- dependent manner, particularly the p-JNK pathway. The effects of VAPs on GRP78 and CHOP were weak. In conclusion, MPP+-induced SH-SY5Y cell death may be linked to ER stress. VAPs prevented MPP+-induced SH-SY5Y cell death by affecting the p-JNK pathway and caspase-12-mediated apoptosis. These findings assist in understanding the mechanism underlying the protective effect of VAPs on neurons.

Application of Nuclear Volume Measurements to Comprehend the Cell Cycle in Root-Knot Nematode-Induced Giant Cells.

Root-knot nematodes induce galls that contain giant-feeding cells harboring multiple enlarged nuclei within the roots of host plants. It is recognized that the cell cycle plays an essential role in the set-up of a peculiar nuclear organization that seemingly steers nematode feeding site induction and development. Functional studies of a large set of cell cycle genes in transgenic lines of the model host Arabidopsis thaliana have contributed to better understand the role of the cell cycle components and their implication in the establishment of functional galls. Mitotic activity mainly occurs during the initial stages of gall development and is followed by an intense endoreduplication phase imperative to produce giant-feeding cells, essential to form vigorous galls. Transgenic lines overexpressing particular cell cycle genes can provoke severe nuclei phenotype changes mainly at later stages of feeding site development. This can result in chaotic nuclear phenotypes affecting their volume. These aberrant nuclear organizations are hampering gall development and nematode maturation. Herein we report on two nuclear volume assessment methods which provide information on the complex changes occurring in nuclei during giant cell development. Although we observed that the data obtained with AMIRA tend to be more detailed than Volumest (Image J), both approaches proved to be highly versatile, allowing to access 3D morphological changes in nuclei of complex tissues and organs. The protocol presented here is based on standard confocal optical sectioning and 3-D image analysis and can be applied to study any volume and shape of cellular organelles in various complex biological specimens. Our results suggest that an increase in giant cell nuclear volume is not solely linked to increasing ploidy levels, but might result from the accumulation of mitotic defects.

Nuclear envelope structural proteins facilitate nuclear shape changes accompanying embryonic differentiation and fidelity of gene expression

BackgroundNuclear size and shape are specific to a cell type, function, and location, and can serve as indicators of disease and development. We previously found that lamin A/C and associated nuclear envelope structural proteins were upregulated when murine embryonic stem (ES) cells differentiated to primitive endoderm cells. Here we further investigated the morphological changes of nuclei that accompany this differentiation.ResultsThe nuclei of undifferentiated wild type cells were found shaped as flattened, irregular ovals, whereas nuclei of Gata4-positive endoderm cells were more spherical, less flattened, and with a slightly reduced volume. The morphological change was confirmed in the trophectoderm and primitive endoderm lineages of E4.5 blastocysts, compared to larger and more irregularly shaped of the nuclei of the inner cell mass. We established ES cells genetically null for the nuclear lamina proteins lamin A/C or the inner nuclear envelope protein emerin, or compound mutant for both lamin A/C and emerin. ES cells deficient in lamin A/C differentiated to endoderm but less efficiently, and the nuclei remained flattened and failed to condense. The size and shape of emerin-deficient nuclei also remained uncondensed after treatment with RA. The emerin/lamin A/C double knockout ES cells failed to differentiate to endoderm cells, though the nuclei condensed but retained a generally flattened ellipsoid shape. Additionally, ES cells deficient for lamin A/C and/or emerin had compromised ability to undergo endoderm differentiation, where the differentiating cells often exhibited coexpression of pluripotent and differentiation markers, such as Oct3/4 and Gata4, respectively, indicating an infidelity of gene regulation.ConclusionsThe results suggest that changes in nuclear size and shape, which are mediated by nuclear envelope structural proteins lamin A/C and/or emerin, also impact gene regulation and lineage differentiation in early embryos. Nevertheless, mice lacking both lamin A/C and emerin were born at the expected frequency, indicating their embryonic development is completed despite the observed protein deficiency.