35S Label Research Articles

Arbuscular mycorrhiza fungi colonisation stimulates uptake of inorganic nitrogen and sulphur but reduces utilisation of organic forms in tomato

Arbuscular mycorrhizal fungi (AMF) form symbioses with most plants, potentially improving their growth and nutrient assimilation activities. Cysteine (Cys) and methionine (Met) are nitrogen (N)- and sulphur (S)-containing amino acids. Compared with phosphate and N, limited attention has been paid to the role of AMF in low molecular weight organic S acquisition. To explore the uptake and relative contributions of organic and inorganic N and S to plants, and the role of AMF in S uptake, a pot study was conducted based on 14C, 35S, 13C, and 15N quad labelling (6-h labelling test after adding the labelled solution to the soil–plant system) using a mutant tomato genotype with highly decreased AMF symbiosis capacity. Tomato roots can uptake limited amounts of added Met and Cys (<1.77%) within 6 h, as indicated by the 14C and 13C labelling results, and most of them were utilised by soil microorganisms. After uptake for 6 h, 10.0–14.8% of N and 1.4–6.1% of S derived from added Cys and Met were utilised by plants, mainly in inorganic N and S forms derived from Cys and Met decomposition. Met and Cys could be important S sources (Met: 3.0–9.8%, Cys: 8.8–22.0%) for plants, however have negligible roles in N nutrition (∼1%), as most N uptake by plants was derived from soil inorganic N. The tomato uptake of inorganic S derived from Cys decomposition was much higher than that derived from Met, as higher ratios of S-Cys were released as SO42− from microorganisms during the 6-h testing periods. Even with the artificial addition of AMF, most of the added Met and Cys were utilised by Gram-negative bacteria, as indicated by 13C-PLFA biomarkers. AMF reduced host plant uptake of organic N and S, but stimulated plant N uptake from Met and Cys, which was mainly inorganic N following mineralisation. AMF not only utilise organic carbon from host plants but also capture soil organic matter to satisfy their energy demands. In the spaces where both root and AMF occur, AMF colonisation decreased tomato 35S uptake from Cys, Met, and SO42− by 24.6%, 20.6%, and 11.0% within the 6-h uptake period, respectively, when compared with that in the mutant genotype with reduced colonisation capacity; in contrast, AMF colonisation increased 35S-Cys uptake by 118.7% from areas that roots could not reach. Overall, AMF enhanced host plant N uptake but reduced organic N uptake under competition with plant roots for S in the rhizosphere, but stimulated plant S uptake by extraradical mycelia, based on this short-term pulse-chase test.

Effects of farmyard manure on soil S cycling: Substrate level exploration of high- and low-molecular weight organic S decomposition

Although S deficiency has been reported in plants worldwide, the belowground biogeochemical cycling of S is not well known. The combined use of mineral fertiliser and manure is regarded as a suitable fertilisation strategy to maintain agricultural soil productivity. A long-term (1964–2018) field experiment was selected to determine how manure application affects soil gross S mineralisation and immobilisation, and plant-derived organic S, cysteine (Cys), and methionine (Met) biological decomposition by 35S, 14C, and 15N labelling. High organic manure application did not increase organic S content in the topsoil owing to the high mineralisation rate, but it increased the organic S content in subsoil where mineralisation rates were relatively lower. S immobilisation dominated gross S fluxes, and the highest SO42− immobilisation rates were recorded under medium manure application. Most plant-derived protein S was decomposed to SO42− after 15 min, and only approximately 30% was retained in the microbial biomass. Protein bioavailability may have a more dominant role in soil S mineralisation than soil S-containing amino acids, owing to its higher concentration. The immobilisation of SO42− was considerably slower than that of proteins and amino acids, which indicates that the microorganisms preferred organic S over inorganic S and that the use is driven by C demand rather than S demand. Moreover, the microbial community released SO42− and NH4+ after taking up Cys and Met, and the imbalance of elements between substrates and microbes played a dominant role in soil S cycling. This process was strongly regulated by the nature of the substrate; less SO42− was released from Met than from Cys. Among the three important processes for organic S decomposition—the uptake by microorganisms, SO42− release, and SO42− reuse—manure application had a greater effect on SO42− release during organic S decomposition. Overall, manure application increased S bioavailability owing to high S fluxes, and high- and low-molecular weight organic S could be rapidly decomposed to SO42−.

Maize and soybean experience fierce competition from soil microorganisms for the uptake of organic and inorganic nitrogen and sulphur: A pot test using 13C, 15N, 14C, and 35S labelling

Nitrogen (N) and sulphur (S) are essential nutrients for plant growth. A pot experiment was conducted to verify whether maize and soybean under monoculture and intercropping could utilise N- and S-containing amino acids when competing with soil microorganisms. Maize and soybean were able to utilise methionine (Met; 1.9–2.2% of total addition) and cysteine (Cys; 0.6–1.6% of total addition) as N and S sources, however they faced competition with soil microorganisms. Six hours after microbial uptake, 45.3–64.0% of the S from Met was retained in the microbial biomass, while there was much lower retention from Cys (16.8–34.5%), and 32.5–44.1% of the S from Cys and 15.6–33.3% of that from Met was transformed to SO42−. Cys was a superior S source for plants compared to Met, as higher SO42− release from Cys could support plant growth. Both maize and soybean plants took up NH4+ (98–99% of total N uptake) and SO42− (85–90%) as their main N and S sources from soil. The N from Cys and Met accounted for only ~1% of total N uptake from soil, and organic Cys and Met accounted for only ~0.2% of the total N uptake, indicating that these two amino acids have limited effects on plant N nutrition, due to the high inorganic N content in agricultural soil. However, the Cys and Met contribution (i.e., organic S uptake and mineral S uptake originating from Cys and Met) to total S uptake (10–15%) was an order of magnitude higher than their N contributions, suggesting that Cys and Met play an important role in soil S nutrition. Intercropping altered the uptake but not the preference for N and S forms in maize and soybean. Overall, the results suggest that S-containing amino acids are important S sources for plant growth even at a lower concentration in soil, but that they play a limited role in plant N nutrition due to a larger inorganic N pool in agricultural soil.

Carbon and sulphur tracing from soil organic sulphur in plants and soil microorganisms

In soils, cysteine and methionine represent the predominant constituents of the low molecular weight organic S, but their role in plant nutrition and soil S cycling is unclear. Cysteine and methionine uptake by pot-cultivated wheat and oilseed rape and their cycling were evaluated using 14C and 35S labelling. About 0.16%–0.30% of 14C from cysteine and methionine was absorbed by the plants after 6 h, indicating that the plants could utilise organic S, with oilseed rape showing higher ability than wheat to take up intact organic S and its derivative inorganic S. Plants utilised organic S with much lower efficiency than inorganic S because most organic S was decomposed by microorganisms. Nitrogen (N) addition enhanced plant S uptake, as high N uptake stimulates S immobilization. About 28%–33% and 67–71% of 14C from cysteine and methionine, respectively, were retained in the microbial biomass (MB), a much higher proportion of 14C from cysteine was released as 14CO2 from the soil than from methionine. Further, 16%–25% and 61%–72% of 35S from cysteine and methionine, respectively, were retained in the MB, and 35%–42% and 5%–9% were released from it as SO42−. Microbial carbon (C) and S use efficiency for cysteine was lower than for methionine, whereas plants utilised higher amounts of cysteine-derived S as sulphate. The higher microbial utilisation rate of methionine by soil microorganisms, compared with cysteine, may reduce the bioavailability of this compound in the soil solution, and, consequently, its uptake by plants.

Single-Site Glycoprotein Mutants Inhibit a Late Event in Sindbis Virus Assembly.

A panel of Sindbis virus mutants that were suspected to have deficiencies in one or more aspects of their replication cycles was examined in baby hamster kidney (BHK) cells. These included an amino acid deletion (ΔH230) and substitution (H230A) in the Sindbis glycoprotein E1_H230 and similar mutants in E2_G209 (G209A, G209D, and ΔG209). Neither H230 mutation produced a measurable titer, but repeated passaging of the H230A mutant in BHK cells produced a second-site compensatory mutant (V231I) that partially rescued both H230 mutants. Electron micrograph (EM) images of these mutants showed assembled viral nucleocapsids but no completed, mature virions. EM of the compensatory mutant strains showed complete virus particles, but these now formed paracrystalline arrays. None of the E2_G209 substitution mutants had any effect on virus production; however, the deletion mutant (ΔG209) showed a very low titer when grown at 37°C and no titer when grown at 28°C. When the deletion mutant grown at 28°C was examined by EM, partially budded virions were observed at the cell surface. (35)S labeling of this mutant confirmed the presence of mutant virus protein in the transfected BHK cell lysate. We conclude that H230 is essential for the assembly of complete infectious Sindbis virus virions and that the presence of an amino acid at E2 position 209 is required for complete budding of Sindbis virus particles although several different amino acids can be at this location without affecting the titer. Our data show the importance of single-site mutations at E1_H230 and E2_G209 in Sindbis virus glycoproteins. These sites have been shown to affect assembly and antibody binding in previous studies. Our data indicate that mutation of one histidine residue in E1 is detrimental to the assembly of Sindbis virus particles in baby hamster kidney cells. Repeated passaging leads to a second-site substitution that partially restores the titer although EM still shows an altered phenotype. Substitutions at position G209 in E2 have no effect on titer, but deletion of this residue greatly reduces titer and again prevents assembly. When this mutant is grown at a lower temperature, virus particles bud from the host cell, but budding arrests before the progeny virus escapes. These results allow us to conclude that these sites have essential roles in assembly, and E2_G209 shows us a new viral egress phenotype.

The Recovery of Plastid Function Is Required for Optimal Response to Low Temperatures in Arabidopsis.

Cold acclimation is an essential response in higher plants to survive freezing temperatures. Here, we report that two independent mutant alleles of the H-subunit of Mg-chelatase, CHLH, gun5-1 and cch in Arabidopsis are sensitive to low temperatures. Plants were grown in photoperiodic conditions and exposed to low temperatures for short- and long-term periods. Tetrapyrrole biosynthesis was initially significantly inhibited in response to low temperature but recovered in wild type (Col-0), although the tetrapyrrole levels were lower in cold compared to control conditions. The gun5-1 and cch alleles showed an inability to recover chlorophyll biosynthesis in addition to a significant decrease in freezing tolerance. We found that the impaired plastid function in the CHLH mutant plants resulted in compromised de novo protein synthesis at low temperatures. The expression of the transcription factors CBF1-3 was super-induced in gun5-1 and cch mutant alleles but expression levels of their target genes, COR15a, COR47 and COR78 were similar or even lower compared to Col-0. In addition, the protein levels of COR15a were lower in gun5-1 and cch and a general defect in protein synthesis could be seen in the gun5-1 mutant following a 35S labelling experiment performed at low temperature. Taken together, our results demonstrate the importance of a functional chloroplast for the cold acclimation process and further suggest that impaired plastid function could result in inhibition of protein synthesis at low temperature.

Tissue and cellular localization of nanoparticles using 35S labeling and light microscopic autoradiography

Microscopical visualization of nanoparticles in tissues is essential for assessing their distribution in whole organisms and their interaction with the cellular microenvironment, including possible toxic effects. However, labeling of nanoparticles with fluorescent dyes may affect their physicochemical properties. Moreover, the detection of organic nanoparticles in their tissue context often poses a particular challenge due to their closer similarities with biomolecules. As part of a biodistribution and toxicity study on organic anti-inflammatory nanoscaled dendritic polyglycerol sulfate amine (dPGS amine) we have established light microscopic autoradiography (LMA) for the tracking of 35S labeled dPGS in standard histopathological tissue samples following intravenous injection in mice. The dPG35S amine was specifically localized in hepatic Kupffer cells with no histopathologic evidence of toxic, degenerate or inflammatory side effects. The combination of radiolabeling of organic nanoparticles with LMA offers a novel approach for their localization in microscopical slides, also allowing for a simultaneous standard toxicopathology analysis. From the Clinical EditorIn this study, a novel light microscopic autoradiography utilizing 35S isotope demonstrates a combined approach to visualize nanoparticle locations in microscopic slides with no obvious toxicity to the studied cells and with minimal external hazard.

Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli

BackgroundBiotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin.ResultsIn this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli.ConclusionsThe strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

Studies on the Non-Protein Thiols of a Human Prostatic Cancer Cell Line: Glutathione Content

The low molecular weight thiol (-SH) content of a human prostate carcinoma cell line (LNCap), important to the cellular resistance to drugs and irradiation, was investigated using three forms of thiol assay each utilizing different chemistries. The composition of the mixture was examined by derivatization of the thiols with a three-fold excess of the Ellman reagent to give mixed aromatic disulfides. The components were isolated by chromatography on C18 reverse phase silica gel followed by DE52 anion exchange separation, and then analyzed by capillary electrophoresis against prepared standards. The glutathione adduct (GSSE) and an unknown disulfide (RSSE) were the major components isolated on DE52 together with two minor ones. However, from the absorbance at 325 nm, it was found that the GSSE isolated (1.5 ± 0.2 femtomoles/cell) could only account for 28.5 ± 4.3% of the total ASF thiols. It appeared that the bulk of the thiol material had not formed a stable mixed disulfide with Ellman’s reagent, and this was confirmed by 35S labeling of the cells. A large proportion of the 35S labeled components, obtained after reaction of the ASF thiols with the Ellman reagent, did not form mixed aromatic disulfides and could therefore not be identified by this labeling method.

Sulfite Reductase Defines a Newly Discovered Bottleneck for Assimilatory Sulfate Reduction and Is Essential for Growth and Development in Arabidopsis thaliana

The role of sulfite reductase (SiR) in assimilatory reduction of inorganic sulfate to sulfide has long been regarded as insignificant for control of flux in this pathway. Two independent Arabidopsis thaliana T-DNA insertion lines (sir1-1 and sir1-2), each with an insertion in the promoter region of SiR, were isolated. sir1-2 seedlings had 14% SiR transcript levels compared with the wild type and were early seedling lethal. sir1-1 seedlings had 44% SiR transcript levels and were viable but strongly retarded in growth. In mature leaves of sir1-1 plants, the levels of SiR transcript, protein, and enzymatic activity ranged between 17 and 28% compared with the wild type. The 28-fold decrease of incorporation of (35)S label into Cys, glutathione, and protein in sir1-1 showed that the decreased activity of SiR generated a severe bottleneck in the assimilatory sulfate reduction pathway. Root sulfate uptake was strongly enhanced, and steady state levels of most of the sulfur-related metabolites, as well as the expression of many primary metabolism genes, were changed in leaves of sir1-1. Hexose and starch contents were decreased, while free amino acids increased. Inorganic carbon, nitrogen, and sulfur composition was also severely altered, demonstrating strong perturbations in metabolism that differed markedly from known sulfate deficiency responses. The results support that SiR is the only gene with this function in the Arabidopsis genome, that optimal activity of SiR is essential for normal growth, and that its downregulation causes severe adaptive reactions of primary and secondary metabolism.

Design and synthesis of sulfur‐35 agents and their applications for protein labeling

AbstractTwo new 35S reagents were developed to radiolabel proteins. The first reagent, N‐succinimidyl‐4‐(methane [35S]sulfonylamino‐methyl)‐benzoate (SMSB), acylates the ε‐amino group of lysine residues in proteins. The second reagent, 4‐(methane [35S]sulfonylamino‐methyl)‐phenylpropylaldehyde (MSAPPA), labels lysine residues via reductive alkylation. Comparing the two methods, the reductive alkylation method labeled proteins over a broader pH range with higher overall radiochemical yield. More than ten monoclonal antibodies (mAbs) have been labeled with these 35S labeling reagents, the biological activity of the mAbs was unchanged. Part of this work was presented in the Ninth International Symposium on the Synthesis and Applications of Isotopically Labelled Compounds, Edinburgh, 16–20 July 2006. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Cinnamon Extract Attenuates TNF-α-induced Intestinal Lipoprotein ApoB48 Overproduction by Regulating Inflammatory, Insulin, and Lipoprotein Pathways in Enterocytes

We have previously reported that the obesity-associated proinflammatory cytokine, TNF-alpha, stimulates the overproduction of intestinal apolipoprotein (apo) B48 containing lipoproteins. In the current study, we have evaluated whether a water-soluble cinnamon extract [CE (Cinnulin PF)] attenuates the dyslipidemia induced by TNF-alpha in Triton WR-1339 treated hamsters, and whether CE inhibits the oversecrection of apoB48-induced by TNF-alpha in enterocytes in a 35S labeling study. In vivo, oral treatment of Cinnulin PF (50 mg per kg BW), inhibited the postprandial overproduction of apoB48-containing lipoproteins and serum triglyceride levels. In ex vivo 35S labeling studies, CE (10 and 20 microg/ml) inhibited the oversecretion of apoB48 induced by TNF-alpha treated enterocytes into the media. To determine the molecular mechanisms, TNF-alpha treated primary enterocytes isolated from chow-fed hamsters, were incubated with CE (10 microg/ml), and the expression of the inflammatory factor genes, IL1-beta, IL-6, and TNF-alpha, insulin signaling pathway genes, insulin receptor (IR), IRS1, IRS2, phosphatidylinositol 3-kinase (PI3-K), Akt1 and phosphatase and tensin homology (PTEN), as well as the key regulators of lipid metabolism, cluster of differentiation (CD)36, microsomal triglyceride transfer protein (MTTP), and sterol regulatory element binding protein (SREBP)-1c were evaluated. Quantitative real-time PCR assays showed that CE treatment decreased the mRNA expression of IL-1beta, IL-6 and TNF-alpha, improved the mRNA expression of IR, IRS1, IRS2, PI3K and Akt1, inhibited CD36, MTTP, and PTEN, and enhanced the impaired SREBP-1c expression in TNF-alpha treated enterocytes. These data suggest that a water extract of cinnamon reverses TNF-alpha-induced overproduction of intestinal apoB48 by regulating gene expression involving inflammatory, insulin, and lipoprotein signaling pathways. In conclusion, Cinulin PF improves inflammation related intestinal dyslipidemia.

Cinnamon extract improves TNFa‐ induced overproduction of intestinal apolipoproteinB 48‐containing lipoproteins

TNFa, stimulates the overproduction of intestinal apolipoproteins. We evaluated whether a water extract of cinnamon (Cinnulin PF®) improved the dyslipidemia induced by TNFa in Triton WR‐1339 treated hamsters, and whether Cinnulin PF® inhibits the TNFa‐induced over secretion of apoB‐48 in hamster enterocytes. In vivo, acute oral intake of Cinnulin PF® (50 mg per kg BW), inhibited the postprandial overproduction of apoB‐48 lipoproteins and serum triglycerides. Ex vivo 35S labeling studies, demonstrated that CE (10 and 20 ?g/mL) inhibited the over secretion of apoB‐48 induced by TNFa treated enterocytes. To determine the effects on the mRNA levels for the related genes of lipoprotein metabolism in enterocytes, fresh isolated primary enterocytes of hamsters were pre‐treated with TNFa(10 ng per mL for 30min), then treated with and without Cinnulin PF® (10ug/mL) for 0.5, 2h and 4h. Quantitative real‐time PCR assays demonstrated that Cinnulin PF® improved the mRNA expression of IR, IRS1, IRS2, PI3K and Akt1and decreased the mRNA expression of MTP, PTEN, IL1a and b, IL6 and TNFa in TNFa treated enterocytes. In summary, our results suggest that a water extract of cinnamon reverses TNFa‐induced overproduction of intestinal apoB‐48 via regulating the gene expression of components of the insulin signaling, lipoprotein and inflammatory pathways. (Funded by USDA/ARS and Integrity Nutraceuticals).

Secreted Cyclophilin A, a Peptidylprolyl cis-trans Isomerase, Mediates Matrix Assembly of Hensin, a Protein Implicated in Epithelial Differentiation

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

SAP18 Promotes Krüppel-dependent Transcriptional Repression by Enhancer-specific Histone Deacetylation

Body pattern formation during early embryogenesis of Drosophila melanogaster relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. We identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation.

Stabilization of N-Myc Is a Critical Function of Aurora A in Human Neuroblastoma

In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. In a shRNA screen of genes that are highly expressed in MYCN-amplified tumors, we have identified AURKA as a gene that is required for the growth of MYCN-amplified neuroblastoma cells but largely dispensable for cells lacking amplified MYCN. Aurora A has a critical function in regulating turnover of the N-Myc protein. Degradation of N-Myc requires sequential phosphorylation by cyclin B/Cdk1 and Gsk3. N-Myc is therefore degraded during mitosis in response to low levels of PI3-kinase activity. Aurora A interacts with both N-Myc and the SCF(Fbxw7) ubiquitin ligase that ubiquitinates N-Myc and counteracts degradation of N-Myc, thereby uncoupling N-Myc stability from growth factor-dependent signals.

Cinnamon extract inhibits the postprandial overproduction of apolipoprotein B48-containing lipoproteins in fructose-fed animals

We have reported previously that a cinnamon extract (CE), high in type A polyphenols, prevents fructose feeding-induced decreases in insulin sensitivity and suggested that improvements of insulin sensitivity by CE were attributable, in part, to enhanced insulin signaling. In this study, we examined the effects of CE on postprandial apolipoprotein (apo) B-48 increase in fructose-fed rats, and the secretion of apoB48 in freshly isolated intestinal enterocytes of fructose-fed hamsters. In an olive oil loading study, a water-soluble CE (Cinnulin PF, 50 mg/kg body weight, orally) decreased serum triglyceride (TG) levels and the over production of total- and TG-rich lipoprotein-apoB48. In ex vivo 35S labeling study, significant decreases were also observed in apoB48 secretion into the media in enterocytes isolated from fructose-fed hamsters. We also investigated the molecular mechanisms of the effects of CE on the expression of genes of the insulin signaling pathway [insulin receptor (IR), IR substrate (IRS)1, IRS2 and Akt1], and lipoprotein metabolism [microsomal TG transfer protein (MTP), sterol regulatory element-binding protein (SREBP1c) in isolated primary enterocytes of fructose-fed hamsters, using quantitative real-time polymerase chain reaction. The CE reversed the expression of the impaired IR, IRS1, IRS2 and Akt1 mRNA levels and inhibited the overexpression of MTP and SREBP1c mRNA levels of enterocytes. Taken together, our data suggest that the postprandial hypertriglycerides and the overproduction of apoB48 can be acutely inhibited by a CE by a mechanism involving improvements of insulin sensitivity of intestinal enterocytes and regulation of MTP and SREBP1c levels. We present both in vivo and ex vivo evidence that a CE improves the postprandial overproduction of intestinal apoB48-containing lipoproteins by ameliorating intestinal insulin resistance and may be beneficial in the control of lipid metabolism.

Inhibition of apoB secretion from HepG2 cells by insulin is amplified by naringenin, independent of the insulin receptor

Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins is characteristic of the dyslipidemia associated with insulin resistance. Recently, we demonstrated that the flavonoid naringenin, like insulin, decreased apoB secretion from HepG2 cells by activation of both the phosphoinositide-3-kinase (PI3-K) pathway and the mitogen-activated protein kinase/extracellular-regulated kinase (MAPK(erk)) pathway. In the present study, we determined whether naringenin-induced signaling required the insulin receptor (IR) and sensitized the cell to the effects of insulin, and whether the kinetics of apoB assembly and secretion in cells exposed to naringenin were similar to those of insulin. Immunoblot analysis revealed that insulin stimulated maximal phosphorylation of IR and IR substrate-1 after 10 min, whereas naringenin did not affect either at any time point up to 60 min. The combination of naringenin and submaximal concentrations of insulin potentiated extracellular-regulated kinase 1/2 activation and enhanced upregulation of the LDL receptor, downregulation of microsomal triglyceride transfer protein expression, and inhibition of apoB-100 secretion. Multicompartmental modeling of apoB pulse-chase studies revealed that attenuation of secreted radiolabeled apoB in naringenin- or insulin-treated cells was similar under lipoprotein-deficient or oleate-stimulated conditions. Naringenin and insulin both stimulated intracellular apoB degradation via a kinetically defined rapid pathway. Therefore, naringenin, like insulin, inhibits apoB secretion through activation of both PI3-K and MAPK(erk) signaling, resulting in similar kinetics of apoB secretion. However, the mechanism for naringenin-induced signaling is independent of the IR. Naringenin represents a possible strategy for reduction of hepatic apoB secretion, particularly in the setting of insulin resistance.

A High-Temporal Resolution Technology for Dynamic Proteomic Analysis Based on 35S Labeling

As more and more research efforts have been attracted to dynamic or differential proteomics, a method with high temporal resolution and high throughput is required. In present study, a 35S in vivo Labeling Analysis for Dynamic Proteomics (SiLAD) was designed and tested by analyzing the dynamic proteome changes in the highly synchronized A549 cells, as well as in the rat liver 2/3 partial hepatectomy surgery. The results validated that SiLAD technique, in combination with 2-Dimensional Electrophoresis, provided a highly sensitivity method to illustrate the non-disturbed endogenous proteins dynamic changes with a good temporal resolution and high signal/noise ratio. A significant number of differential proteins can be discovered or re-categorized by this technique. Another unique feature of SiLAD is its capability of quantifying the rate of protein expression, which reflects the cellular physiological turn points more effectively. Finally, the prescribed SiLAD proteome snapshot pattern could be potentially used as an exclusive symbol for characterizing each stage in well regulated biological processes.

Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1α

We have reported previously that PX-478 (S-2-amino-3-[4'-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1alpha (HIF-1alpha) within the tumor. We now report that PX-478 inhibits HIF-1alpha protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1alpha inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1alpha mRNA and inhibits translation as determined by 35S labeling experiments and reporter assays using the 5' untranslated region of HIF-1alpha. Moreover, to a lesser extent, PX-478 also inhibits HIF-1alpha deubiquitination resulting in increased levels of polyubiquitinated HIF-1alpha. The inhibitory effect of PX-478 on HIF-1alpha levels is primarily due to its inhibition of translation because HIF-1alpha translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1alpha at multiple levels that together or individually may contribute to its antitumor activity against HIF-1alpha-expressing tumors.