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BACKGROUND: Nanotechnology has been widely applied in agricultural science. During the process of reducing metal toxicity and accumulation in rice, nanomaterials exhibit size effects. However, there is limited knowledge regarding these size effects. We aim to explore the impact of fertilizer with various sizes of ZnO nanoparticles (ZnO‐NPs) on rice growth and cadmium (Cd) accumulation and to elucidate the potential mechanism of Cd reduction in rice. Foliar applications of different concentrations (0.5 and 2 mmol L−1) and different sizes (30 and 300 nm ZnO‐NPs) of zinc (Zn) fertilizer (Zn(NO3)2) were performed to investigate the effects on rice growth, Cd accumulation and subcellular distribution, and the expression of Zn–Cd transport genes. RESULTS: The results suggested that all the foliar sprayings can significantly reduce the Cd concentrations in rice grains by 41–61% with the highest reduction in the application of ZnO‐NPs with large size and low concentration. This is related to the enhancement of Cd fixation in leaf cell walls and downregulation of Cd transport genes (OsZIP7, OsHMA2, OsHMA3) in stem nodes. Foliar ZnO‐NPs applications can increase the Zn concentration in grains by 9–21%. Foliar applications of Zn(NO3)2 and small‐sized ZnO‐NPs promoted plant growth and rice yield, while the application of large‐sized ZnO‐NPs significantly reduced rice growth and yield. CONCLUSION: The study suggests that the rice yield and Cd reduction are dependent on the size and concentration of foliar spraying and the use of large‐sized ZnO‐NPs is the most effective strategy when considering both yield and Cd reduction comprehensively. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Characterization of the subcellular distribution of RNA is essential for understanding the molecular basis of biological processes. Here, the subcellular nanopore direct RNA‐sequencing (DRS) of four lung cancer cell lines (A549, H1975, H358, and HCC4006) is performed, coupled with a computational pipeline, Low‐abundance Aware Full‐length Isoform clusTEr (LAFITE), to comprehensively analyze the full‐length cytoplasmic and nuclear transcriptome. Using additional DRS and orthogonal data sets, it is shown that LAFITE outperforms current methods for detecting full‐length transcripts, particularly for low‐abundance isoforms that are usually overlooked due to poor read coverage. Experimental validation of six novel isoforms exclusively identified by LAFITE further confirms the reliability of this pipeline. By applying LAFITE to subcellular DRS data, the complexity of the nuclear transcriptome is revealed in terms of isoform diversity, 3'‐UTR usage, m6A modification patterns, and intron retention. Overall, LAFITE provides enhanced full‐length isoform identification and enables a high‐resolution view of the RNA landscape at the isoform level. [ABSTRACT FROM AUTHOR]

Abstract Characterization of the subcellular distribution of RNA is essential for understanding the molecular basis of biological processes. Here, the subcellular nanopore direct RNA‐sequencing (DRS) of four lung cancer cell lines (A549, H1975, H358, and HCC4006) is performed, coupled with a computational pipeline, Low‐abundance Aware Full‐length Isoform clusTEr (LAFITE), to comprehensively analyze the full‐length cytoplasmic and nuclear transcriptome. Using additional DRS and orthogonal data sets, it is shown that LAFITE outperforms current methods for detecting full‐length transcripts, particularly for low‐abundance isoforms that are usually overlooked due to poor read coverage. Experimental validation of six novel isoforms exclusively identified by LAFITE further confirms the reliability of this pipeline. By applying LAFITE to subcellular DRS data, the complexity of the nuclear transcriptome is revealed in terms of isoform diversity, 3'‐UTR usage, m6A modification patterns, and intron retention. Overall, LAFITE provides enhanced full‐length isoform identification and enables a high‐resolution view of the RNA landscape at the isoform level.

This study aimed to determine the role of sulfhydryl compounds in the subcells of C. rupestris under Pb stress. Different concentrations (0, 0.5, 1.0, 2.5, 5.0, 7.5, and 10 mg/L) and different exposure days (1, 3, 5, and 7 days) were designed to analyze the subcellular distribution of non-protein thiols (NPT), glutathione (GSH), and phytochelatins (PCs) in C. rupestris. NPT, GSH, and PCs increased significantly with increasing Pb stress in the cell wall and soluble fraction, especially NPT. NPT and GSH slowly increased, and PCs showed no significant difference in the organelle of C. rupestris at low concentrations (< 5.0 mg/L). PCs slightly increased under 5.0 mg/L of Pb stress. PCs/NPT gradually increased with Pb stress at a low Pb concentration. GSH detoxification response lagged behind those of NPT and PCs in response to time. PCs/NPT initially increased and then decreased with Pb stress duration. This study suggested that NPT, GSH, and PCs played an important role in the detoxification of the cell wall and the soluble fraction of C. rupestris under Pb stress. PCs were important in the organelle. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the endogenous cathepsin activity in each subcellular fraction and the effect of this activity on myofibrillar protein and texture during refrigeration and partial freezing storage of northern pike (Esox lucius) fillets. The results showed that fillets stored under the refrigerated condition were more susceptible to oxidation than partial freezing. Endogenous cathepsin activity indicated that partial freezing destroys the integrity of lysosomes more effectively than refrigeration and inhibits the increase in cathepsin B and B + L in lysosomes. The activity of cathepsin B and B + L in lysosomes, mitochondria and myofibrils under the partial freezing conditions was always lower than that under refrigeration. Texture analysis showed that refrigeration had a negative impact on hardness and springiness. In conclusion, the cathepsin activity in each subcellular fraction was effectively inhibited and better textural characteristics were obtained with partial freezing than refrigeration. [ABSTRACT FROM AUTHOR]

Understanding the precise intracellular localization of lead (Pb) is a key in deciphering processes in Pb-induced toxicology. However, it is a great challenge to trace Pb in vitro, especially in cultured cells. We aimed to find an innovative and efficient approach to investigate distribution of Pb in cells and to validate it through determining the subcellular Pb content. We identified its ultra-structural distribution with autometallography under electron microscopy in a choroidal epithelial Z310 cell line. Electron microscopy in combination with energy-dispersive X-ray spectroscope (EDS) was employed to provide further evidence of Pb location. In addition, Pb content was determined in the cytosol, membrane/organelle, nucleus and cytoskeleton fractions with atomic absorption spectroscopy. Pb was found predominantly inside the nuclear membranes and some was distributed in the cytoplasm under low-concentration exposure. Nuclear existence of Pb was verified by EDS under electron microscopy. Once standardized for protein content, Pb percentage in the nucleus fraction reached the highest level (76%). Our results indicate that Pb is accumulated mainly in the nucleus of choroid plexus. This method is sensitive and precise in providing optimal means to study the ultra-structural localization of Pb for in vitro models. In addition, it offers the possibility of localization of other metals in cultured cells. Some procedures may also be adopted to detect target proteins via immunoreactions. [ABSTRACT FROM AUTHOR]

The spatial localization of RNA within cells is closely related to its function and also involved in cell fate determination. However, the atlas of RNA distribution within cells and dynamic changes during the developmental process are largely unknown. In this study, five subcellular components, including cytoplasmic extract, membrane extract, soluble nuclear extract, chromatin-bound nuclear extract, and cytoskeletal extract, were isolated and the rules of subcellular RNA distribution in human embryonic stem cells (hESCs) and its change during hESC differentiation are summarized for the first time. The overall distribution patterns of coding and non-coding RNAs are revealed. Interestingly, some developmental genes are found to be transcribed but confined to the chromatin in undifferentiated hESC. Unexpectedly, alternative splicing and polyadenylation endow spatial heterogeneity among different isoforms of the same gene. Finally, the dynamic pattern of RNA distribution during hESC differentiation is characterized, which provides new clues for a comprehensive understanding hESC pluripotency and differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract Cadmium (Cd) and benzo [ a ]pyrene (BaP) often co-occur in the environment, and the critical body residue of organisms is used as an indicator of the toxic effects of contaminants. However, little is known about their distributions and toxicities when pollution of Cd and BaP are combined. Semi-static solution culture experiment was used to study the impacts of BaP on the subcellular distribution of the toxic metal Cd in the earthworm Eisenia fetida. We explored the mechanisms by which this organism responds to combined exposure to these pollutants by measuring the protein content of each of three subcellular fractions, as well as acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities. The subcellular partitioning of Cd was heterogeneous and Cd mainly accumulated in the cytosolic fraction (Fraction C), which was previously reported to be involved in metal immobilization. In Fraction C, Cd accumulation was correlated with the external concentration to which the earthworm had been exposed; however, in the presence of BaP, Cd accumulation was inhibited and plateaued at high external Cd concentrations. A principal component analysis revealed that this decreased Cd accumulation might be caused by increases in GST activity, which likely increased the excretion of Cd. BaP was also found to stimulate protein biosynthesis and upregulate AChE and GST activities in the debris fraction (Fraction E), indicating other potential detoxification mechanisms in this fraction. Granule fraction (Fraction D) had a lower protein content, AChE and GST activities than the other subcellular fractions, supporting previous findings that Fraction D is largely inert. Graphical abstract Image 1 Highlights • Cadmium (Cd) subcellular partitioning is heterogeneous. • Benzo [ a ]pyrene (BaP) inhibits Cd accumulation. • BaP promoted the production of total protein in the cellular debris fraction. • Glutathione S-transferase (GST) is the main influencer of Cd accumulation. [ABSTRACT FROM AUTHOR]

Zymographic analysis showed that all subcellular fractions of the serogroups possessed different degree of lipolytical activity. Maximal hydrolysis in the radial enzyme-diffusion reaction in agarose gel was observed in Tween-20, Tween-80, Triton Х-305 and Triton Х-405. Total hydrolase activity of urea extracts in Tween-20, Tween-80, Triton Х-405, Span-85 excelled those in the preparations of outer membranes. Maximal lipolytical activity in regard to the Tweens was shown by the preparations of urea extracts prepared from the nontoxigenic strains of O1 serogroups, while in relation to the Tritons - preparations of urea extracts from the toxigenic strains (p < 0,05).

Marine herbivorous fish directly consume macroalgae, which commonly accumulate high levels of trace metals in polluted areas. We proposed that herbivorous fish could be better candidates for biomonitoring marine metal pollution than carnivorous fish. To date, the trophic transfer of Cu from macroalgae to marine herbivorous fish is unclear. In this study, the kinetics of Cu bioaccumulation in a widespread marine herbivorous fish, Siganus oramin, were investigated, and biokinetic modeling was applied to estimate the Cu levels in the fish sampled from different sites and seasons. The results showed that Cu accumulation in the fish was linearly correlated to the dietary Cu levels in the different prey species, which were proportional to the waterborne Cu concentrations. The Cu found in the subcellular trophically available metal fraction (TAM) in the prey contributed the largest proportion of accumulated Cu in S. oramin . The dietary assimilation efficiencies (AEs) of Cu were 15.56 ± 1.76%, 13.42 ± 2.86%, and 21.36 ± 1.47% for Ulva lactuca , Gracilaria lemaneiformis and Gracilaria gigas, respectively. The calculated waterborne uptake rate constant ( k u ) of Cu was 0.023 ± 0.011 L g −1  d −1 , and the efflux rate constant ( k e ) was 0.055 ± 0.021 d −1 . Dietary Cu accounted for 60%–75% of the body Cu in S. oramin , suggesting that dietary uptake could be the primary route for Cu bioaccumulation in herbivorous fish. The biokinetic model demonstrated that the Cu concentrations in the water and fish presented a positive linear relationship, which was in line with our field investigation along the coastal areas of South China. Therefore, we suggested that S. oramin could be used as a biomonitoring organism for Cu pollution in the marine environment. However, the heterogeneities between the predicted levels and the measured levels of Cu implied that seasonal changes should be taken into account to improve the accuracy of the model. [ABSTRACT FROM AUTHOR]

Polygonum hydropiper is a dominant plant species in Shifang phosphorus (P) mine area and is a promising P-accumulator used for P-phytoextraction. To date, little information is available on the physiological response involved in alleviating P toxicity of P. hydropiper under high P. A pot experiment was carried out to investigate growth, P subcellular distribution, chemical forms in two ecotypes of P. hydropiper under high levels (1, 4, and 8 mmol P L −1 ) of inorganic P (Pi) and organic P (Po), supplied as KH 2 PO 4 and myo -inositol hexaphosphoric acid dodecasodium salt, respectively. The mining ecotype (ME) showed a greater ability to tolerate high P than the non-mining ecotype (NME), as shown by its superior growth with undamaged leaf anatomical structure. The ME showed 1.3–2.2 times greater shoot P accumulation than the NME. More than 93% of P accumulated in tissue cell wall and soluble fraction. The increasing P treatments increased all tissue P forms, especially Pi form. The ME showed significantly higher ester P, nucleic P and insoluble P in tissues than the NME at 8 mmol L −1 ; however, it demonstrated lower Pi, expect for roots at 5 weeks. The percentages of Pi and nucleic P in roots of the ME were higher than other P forms, and the percentages of nucleic P dominated in the leaves. Probably, the combination of preferential distribution of P in cell wall and soluble fraction in tissues and storage of P in low activity as nucleic P in leaves allows the ME to adapt high P. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) absorption and accumulation vary greatly not only among plant species but also among cultivars within the same species. In order to better understand the mechanisms of Cd absorption, transportation and distribution, we examined the differences of Cd absorption, translocation, subcellular distribution and chemical forms between L19, a Cd-tolerant genotype, and H4, a Cd-sensitive genotype, using kinetic analysis and soil culture experiment. Kinetic assays showed that the different Cd concentrations between the two cultivars might be ascribed to root absorption and translocation from root to shoot. The investigations of subcellular distribution and chemical forms verified that Cd concentrations of all subcellular fractions in H4 were all higher than in L19. Meanwhile, most of the Cd was associated with cell walls in the root of H4, but the Cd in the root of L19 and leaf of the two cultivars was mainly stored in soluble fraction, which could be one possible mechanism of tolerance to Cd toxicity. In addition, Cd fractions extracted by 1 M NaCl and 2% HAC were predominant in root and leaf of both cultivars and the concentrations and proportions extracted by water and 80% ethanol in root and 1 M NaCl in leaf were all higher in H4 than in L19. These results indicate that the Cd in H4 is more active than L19, which could be responsible for the sensitivity of H4 to Cd damage. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Agricultural Science & Technology (1008-0864) is the property of Journal of Agricultural Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Mitochondria are essential for several biological processes including energy metabolism and cell survival. Accordingly, impaired mitochondrial function is involved in a wide range of human pathologies including diabetes, cancer, cardiovascular, and neurodegenerative diseases. Within the past decade a growing body of evidence indicates that reversible phosphorylation plays an important role in the regulation of a variety of mitochondrial processes as well as tissue-specific mitochondrial functions in mammals. The rapidly increasing number of mitochondrial phosphorylation sites and phosphoproteins identified is largely ascribed to recent advances in phosphoproteomic technologies such as fractionation, phosphopeptide enrichment, and high-sensitivity mass spectrometry. However, the functional importance and the specific kinases and phosphatases involved have yet to be determined for the majority of these mitochondrial phosphorylation sites. This review summarizes the progress in establishing the mammalian mitochondrial phosphoproteome and the technical challenges encountered while characterizing it, with a particular focus on large-scale phosphoproteomic studies of mitochondria from human skeletal muscle. [ABSTRACT FROM AUTHOR]

Ezrin and Annexin seven (A7) have been suggested to be involved in several roles in cancers metastasis. However, the role of Ezrin and the effect of A7 on Ezrin expression in lymphatic metastatic hepatocellular carcinoma (LNM-HCC) have not been extensively explored yet. This study reports expression of Ezrin in high lymphatic metastasis (Hca-F >70%) and low metastatic metastasis (Hca-P <30%) HCC cell lines, and the effect of A7 on Ezrin expression. Real-Time PCR, Western blot, Subcellular fractionation, Immunocytochemistry and Immunofluorescence were used to investigate Ezrin expression in addition to migration and invasion behaviors of A7 up-regulated Hca-F cells, A7 down-regulated Hca-P and in their respective negative control (NC) cells. Ezrin expression was higher in high LNM-HCC than low LNM-HCC ( p = 0.0046). Cell fractionation analysis reveals that Ezrin was highly present in the cytoplasm, nucleus and cytoskeleton of NC-Hca-F cells. However, Ezrin was highly observed in the cell membrane, nucleus and cytoskeleton of NC-Hca-P cells. A7 up-regulation in Hca-F suppressed Ezrin expression ( p = 0.0248), but increase the migration and invasion, whereas Ezrin was mainly located in the cytoplasm and nucleus fractions. Down-regulation of A7 in Hca-P cells, enhanced Ezrin expression ( p < 0.0001) in the cytoplasm and nucleus fractions, and suppressed migration and invasion. In conclusion, Ezrin may play a role in LNM-HCC and might be inversely associated with A7 expression. The subcellular localization of Ezrin and A7 was varied according to the metastatic levels. Ezrin may thus be a potential diagnostic and/or prognostic biomarker for HCC. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Agro-Environment Science is the property of Journal of Agro-Environment Science Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

With the use of diffusion test in substrate agarose gel in sterile subcellular fractions of Vibrio cholerae 01 and 0139 serogroups, got from by the lysis urea of living microbial cells, found out a ability for the proteolysis of rabbit and human immunoglobulins of class G. Substrate electrophoresis detected polypeptides with IgG-protease activity both in water-soluble and membrane fractions of the strains with different molecular masses. Differences in IgG-protease activity of subcellular fractions of V. cholerae ompT+ and ompT-strains were determined.

The potential uptake and trophic transfer ability of nanoparticles (NPs) in aquatic organisms have not been well understood yet. There has been an increasing awareness of the subcellular fate of NPs in organisms, but how the subcellular distribution of NPs subsequently affects the trophic transfer to predator remains to be answered. In the present study, the food chain from Scenedesmus obliquus to Daphnia magna was established to simulate the trophic transfer of fullerene aqueous suspension ( n C 60 ). The n C 60 contaminated algae were separated into three fractions: cell wall (CW), cell organelle (CO), and cell membrane (CM) fractions, and we investigated the n C 60 uptake amounts and trophic transfer efficiency to the predator through dietary exposure to algae or algal subcellular fractions. The n C 60 distribution in CW fraction of S. obliquus was the highest, following by CO and CM fractions. n C 60 uptake amounts in D. magna were found to be mainly relative to the NPs’ distribution in CW fraction and daphnia uptake ability from CW fraction, whereas the n C 60 trophic transfer efficiency (TE) were mainly in accordance with the transfer ability of NPs from the CO fraction. CW fed group possessed the highest uptake amount, followed by CO and CM fed groups, but the presence of humic acid (HA) significantly decreased the n C 60 uptake from CW fed group. The CO fed groups acquired high TE values for n C 60 , while CM fed groups had low TE values. Moreover, even though CW fed group had a high TE value; it decreased significantly with the presence of HA. This study contributes to the understanding of fullerene NPs’ dietary exposure to aquatic organisms, suggesting that NPs in different food forms are not necessarily equally trophically available to the predator. [ABSTRACT FROM AUTHOR]

Cereal grain outer layers fulfil essential functions for the developing seed such as supplying energy and providing protection. In the food industry, the grain outer layers called 'the bran' is valuable since it is rich in dietary fibre and other beneficial nutriments. The outer layers comprise several tissues with a high content in cell wall material. The cell wall composition of the grain peripheral tissues was investigated with specific probes at a stage of active cell wall synthesis. Considerable wall diversity between cell types was revealed. To identify the cellular machinery involved in cell wall synthesis, a subcellular proteomic approach was used targeting the Golgi apparatus where most cell wall polysaccharides are synthesized. The tissues were dissected into outer pericarp and intermediate layers where 822 and 1304 proteins were identified respectively. Many carbohydrate-active enzymes were revealed: some in the two peripheral grain fractions, others only in one tissue. Several protein families specific to one fraction and with characterized homologs in other species might be related to the specific detection of a polysaccharide in a particular cell layer. This report provides new information on grain cell walls and its biosynthesis in the valuable outer tissues, which are poorly studied so far. A better understanding of the mechanisms controlling cell wall composition could help to improve several quality traits of cereal products (e.g. dietary fibre content, biomass conversion to biofuel). [ABSTRACT FROM AUTHOR]

Abstract: Plant cell walls are complex structures critical for plant fitness and valuable for human nutrition as dietary fiber and for industrial uses such as biofuel production. The cell wall polysaccharides in wheat endosperm consist of two major polymers, arabinoxylans and beta-glucans, as well as other minor components. Most of these polysaccharides are synthesized in the Golgi apparatus but the mechanisms underlying their synthesis have yet to be fully elucidated and only a few of the enzymes involved have been characterized. To identify actors involved in the wheat endosperm cell wall formation, we used a subcellular fractionation strategy to isolate Golgi-enriched fractions from endosperm harvested during active cell wall deposition. The proteins extracted from these Golgi-enriched fractions were analyzed by LC–MS/MS. We report the identification of 1135 proteins among which 64 glycosyltransferases distributed in 17 families. Their potential function in cell wall synthesis is discussed. In addition, we identified 63 glycosylhydrolases, some of which may be involved in cell wall remodeling. Several glycosyltransferases were validated by showing that when expressed as fusion proteins with a fluorescent reporter, they indeed accumulate in the Golgi apparatus. Our results provide new candidates potentially involved in cell wall biogenesis in wheat endosperm. [Copyright &y& Elsevier]

Abstract: Several man-made organic pollutants including polychlorinated biphenyls (PCBs) and several pesticides may exhibit endocrine disrupting (ED) properties. These ED molecules can be comparatively persistent in the environment, and have shown to perturb hormonal activity and several physiological functions. The objective of this investigation was to study the impact of PCB 153 and atrazine on human MCF-7 cells, and to search for marker proteins of their exposure. Cells were exposed to environmentally high but relevant concentrations of atrazine (200ppb), PCB 153 (500ppb), 17-β estradiol (positive control, 10nM) and DMSO (0.1%, negative control) for t=36h (n=3 replicates/exposure group). Proteins from cell membrane and cytosol were isolated, and studied by 2D-DiGE. Differentially regulated proteins were trypsin-digested and identified by MALDI-ToF-ToF and NCBInr database. A total of 36 differentially regulated proteins (>|1.5| fold change, P <0.05) were identified in the membrane fraction and 22 in the cytosol, and were mainly involved in cell structure and in stress response, but also in xenobiotic metabolism. 67% (membrane) and 50% (cytosol) of differentially regulated proteins were more abundant following atrazine exposure whereas nearly 100% (membrane) and 45% (cytosol) were less abundant following PCB 153 exposure. Western blots of selected proteins (HSBP1, FKBP4, STMN1) confirmed 2D-DiGE results. This study emphasizes the numerous potential effects that ED compounds could have on exposed humans. [Copyright &y& Elsevier]

Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I – representatives of IA, IB, and IC families, respectively – was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated. Further, evidence is presented that HsdR is phosphorylated in vivo only when coproduced with HsdM and HsdS subunits – as part of assembled EcoKI restriction endonuclease, while the individually produced HsdR subunit is not phosphorylated. In vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of a Type I R-M enzyme reported. [ABSTRACT FROM AUTHOR]

Proteomics combined with cell fractionation was used to identify proteins regulated by high glucose (HG) in human mesangial cells (HMC). Total membrane and cytosolic fraction proteins derived from HMC after 7 days of HG exposure were resolved by a two-dimensional gel electrophoresis approach. DeCyder software was used to analyze the HG-induced protein spot dysregulation. In the membrane subproteome, of the 92 spots that were matched across all gels, HG induced significant downregulation of only 4 protein spots. The dysregulated spots from the membrane subproteome included binding protein (BiP), calreticulin precursor protein, a 63-kDa transmembrane protein from a ER/Golgi intermediate, and 3-subunit Of collagen proline 4-hydroxylase. In the cytosolic subproteome, of the 122 spots that were matched across all gels, HG induced downregulation of 3 protein spots and upregulation of 2 protein spots significantly. Enolase 1, annexin VI, and -γ2-actin were decreased, whereas heat shock protein-70 kDa and calmodulin (CaM) were increased. Further confocal microscopy and Western immunoblotting of mesangial cells validated the increase in CaM. Immunoblotting of diabetic mouse and rat kidneys exhibited a marked increase in CaM at both early and late stages of diabetes, reflecting the potential physiological relevance of CaM upregulation. CaM-specific inhibitors blocked glucose transport stimulated by transforming growth factor-β and insulin in mesangial cells. In conclusion, using a combination of cell fractionation and protein expression profiling, we identified a cohort of HG-dysregulated proteins in the HMC and identified a critical and as yet unrecognized role for CaM in glucose transport in mesangial cells. [ABSTRACT FROM AUTHOR]

Abstract: Seleno-glutathione peroxidases are an important family of antioxidant enzymes, that include the phospholipid hydroperoxide glutathione peroxidase (GPx-4), an enzyme that reduces lipid hydroperoxides in membranes. The essential characteristics of platelet GPx-4 were found to be the same as the GPx-4 from other tissues. To explore the subcellular expression of GPx-4 in human platelets, we first investigated both its activity and localization in subcellular fractions. About 47% of the total cell enzyme activity was found in the membrane fractions, 29% in the mitochondria and 23% in the cytosol fractions. The same subcellular distribution of GPx-4 protein was demonstrated in resting platelets. This distribution data was further established by confocal microscopy. Of major potential biological significance, this distribution changed when platelets were activated. Confocal immunofluorescence microscopy localized mainly GPx-4 to membranes in contrast to cytoplasm in the resting cells. Based on these results we propose that cytoplasmic GPx-4 could be moved to the membrane for protection during platelet activation. This enzyme would then be important to maintain the integrity of platelet function in vascular system stressed by oxidative reactions. [Copyright &y& Elsevier]

Liver, as an important metabolic and detoxicological organ of human body, can be used as a good bioindicator for evaluating body burden of environmental pollutants. Its elemental contents and their chemical forms are closely related to the status of human health and disease. In this paper, the liver samples collected from normal subjects were separated to different subcellular fractions of nuclei, mitochondria, lysosome, microsome and cytosol by differential centrifugation. Then their concentrations of heavy metals of As, Pb, Cd, and Hg were determined by atomic absorption and atomic fluorescent spectroscopy. Our results show no significant difference with literature ones when comparing their gross concentrations. In the case of their subcellular distribution, the Hg concentrations are higher in mitochondrial, microsomal and cytosolic fractions; the Cd concentrations are higher in cytosolic and mitochondrial fractions, while As highest in nuclear fraction. The highest concentration of Pb is found in microsomal fraction with similarity to Fe. Mercury in liver is mainly in the form of inorganic, and methylmercury ranged from 9% to 50% with the average value of 20.9% ±13.3 %. These results indicate that the cellular distribution and the accumulated target organelles are quite different among these heavy metals, which suggest their various pathways and toxic mechanism in vivo. [ABSTRACT FROM AUTHOR]

Cellular localization of Type I restriction–modification enzymes EcoKI, EcoAI, and EcoR124I—the most frequently studied representatives of IA, IB, and IC families—was analyzed by immunoblotting of subcellular fractions isolated from Escherichia coli strains harboring the corresponding hsd genes. EcoR124I shows characteristics similar to those of EcoKI. The complex enzymes are associated with the cytoplasmic membrane via DNA interaction as documented by the release of the Hsd subunits from the membrane into the soluble fraction following benzonase treatment. HsdR subunits of the membrane-bound enzymes EcoKI and EcoR124I are accessible, though to a different extent, at the external surface of cytoplasmic membrane as shown by trypsinization of intact spheroplasts. EcoAI strongly differs from EcoKI and EcoR124I, since neither benzonase nor trypsin affects its association with the cytoplasmic membrane. Possible reasons for such a different organization are discussed in relation of the control of the restriction–modification activities in vivo. [Copyright &y& Elsevier]

Ceramides (Cer) are key intermediates in the metabolism of sphingomyelin and are also important second messengers. We report that natural long-chain ceramides added to the incubation medium in microgram amounts are internalized in HL-60 cells as well as the short-chain analogue C2-Cer and targeted to various subcellular compartments. No significant difference was detected in the ability of HL-60 cells to metabolize exogenous Cer containing a short (acetyl) versus long (palmitoyl or oleoyl) acyl chain. After a 2-h incubation time with [14C]-C16 ceramides, most of the cell-bound radioactivity was found in free ceramides. Sphingomyelin was the major metabolized sphingolipid containing labeled ceramides and only a small proportion of exogenous ceramides were converted to neutral glycolipids and gangliosides. Up to 20% of the exogenous ceramides taken up by the cells were recovered in mitochondria, mostly as authentic C16 ceramides and C16 sphingomyelin, along with a trace amount of labeled GM3 ganglioside. These results are consistent with the notion that exogenous natural ceramides enter cells, can be further metabolized in situ and partly targeted to mitochondria, which are known to be involved in the control of programmed cell death. [Copyright &y& Elsevier]

The low- and high-arsenic (As) transferring cultivars (LTC and HTC) of cabbage showed significant differences in As uptake and distribution. We hypothesise that chemistry of root cell wall matrix plays a critical role. LTC and HTC were treated with As and grown for 60 days. As concentration and distribution at subcellular and cell wall component (pectin, hemicellulose and lignin) levels were determined. Remodelling enzymes (PME and PAL) and functional groups of cell wall were analysed. Results showed that shoot biomass of LTC was not affected by As. Less As was accumulated in shoot of LTC than HTC. LTC allocated more As in root and majority of As was deposited in cell wall. LTC had more hemicellulose 1 (HC1) and lignin, PME and PAL activities. The uronic acid contents of pectin, HC1 or HC2 were all positively (P < 0.05) correlated with As concentrations in each component, respectively. Chemistry of LTC root cell wall was remodelled in terms of changes in porosity, HC and lignin contents, and functional groups, which potentially exerted coupling effects on As entering and deposition. The LTC can restrain As in roots through changing characteristics of root cell wall matrix. [Display omitted] • Low- and high-arsenic (As) transferring cabbages (LTC and HTC) varied in As uptake. • More As was deposited in root cell wall of LTC than HTC treated with As. • LTC had more hemicellulose (HC), lignin and cell wall remodelling enzymes than HTC. • As was mainly associated with pectin and hemicellulose in cell wall. • Pectin, HC, lignin and functional groups induced coupling effects on As fixation. [ABSTRACT FROM AUTHOR]

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl/Ca independent [H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl dependent/Ca activated [H]glutamate binding sites were more abundant and exhibited a single K in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 and trans-ACPD inhibited [H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere. Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [H]AMPA binding. The density of the high affinity [H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [H]kainate binding sites. The density of the latter sites is close to that of the high affinity [H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network. [ABSTRACT FROM AUTHOR]