Sucrose Density Gradient Centrifugation Research Articles

Bacterial heavy metal transporter MerC increases mercury accumulation in Arabidopsis thaliana

This study evaluated the feasibility of transgenic Arabidopsis engineered to express the bacterial heavy metal transporter MerC for the phytoremediation of mercury pollution. MerC, MerC–SYP121, or MerC–AtVAM3 proteins were found to be expressed in leaf segments of transgenic plants using an anti-MerC antibody immunostaining method. By sucrose density gradient centrifugation and immunoblotting analyses, MerC, MerC–SYP121, and MerC–AtVAM3 were found to localized in the Golgi apparatus, plasma membrane, and vacuole membrane, respectively. Transgenic Arabidopsis plants that expressed merC–SYP121 were more resistant to mercury and accumulated significantly more of this metal than wild-type Arabidopsis. These results demonstrated that expression of the bacterial heavy metal transporter MerC promoted the transport and accumulation of mercury in transgenic Arabidopsis, which may be a useful method for improving plants for the phytoremediation of mercury pollution.

Arabidopsis ABCB21 is a Facultative Auxin Importer/Exporter Regulated by Cytoplasmic Auxin Concentration

The phytohormone auxin is critical for plant growth and many developmental processes. Members of the P-glycoprotein (PGP/ABCB) subfamily of ATP-binding cassette (ABC) transporters have been shown to function in the polar movement of auxin by transporting auxin over the plasma membrane in both monocots and dicots. Here, we characterize a new Arabidopsis member of the ABCB subfamily, ABCB21/PGP21, a close homolog of ABCB4, for which conflicting transport directionalities have been reported. ABCB21 is strongly expressed in the abaxial side of cotyledons and in junctions of lateral organs in the aerial part, whereas in roots it is specifically expressed in pericycle cells. Membrane fractionation by sucrose density gradient centrifugation followed by Western blot showed that ABCB21 is a plasma membrane-localized ABC transporter. A transport assay with Arabidopsis protoplasts suggested that ABCB21 was involved in IAA transport in an outward direction, while naphthalene acetic acid (NAA) was a less preferable substrate for ABCB21. Further functional analysis of ABCB21 using yeast import and export assays showed that ABCB21 mediates the 1-N-naphthylphthalamic acid (NPA)-sensitive translocation of auxin in an inward direction when the cytoplasmic IAA concentration is low, whereas this transporter mediates outward transport under high internal IAA. An increase in the cytoplasmic IAA concentration by pre-loading of IAA into yeast cells abolished the IAA uptake activity by ABCB21 as well as ABCB4. These findings suggest that ABCB21 functions as a facultative importer/exporter controlling auxin concentrations in plant cells.

Dissecting the sub-structure of the intraflagellar transport complex B

The intraflagellar transport (IFT) machinery is composed of mainly of two major components, IFT complex A and B as well as of motor proteins (kinesins and dyneins). The aim of this study was to identify comprehensively the composition of the IFT complex B as well as the structure of its functional sub-modules. We applied Strep/FLAG-tandem affinity purification (SF-TAP) and yeast-two-hybrid to identify protein complexes and protein-protein interactions within IFT complex B. By combining these methods with the biochemical separation and destabilization of the protein complex B into sub-complexes and mass spectrometric analysis, we further determined its structure. As a first step, we comprehensively identified the composition of the IFT complex B by SF-TAP using several IFT complex B proteins as baits. The sub-complex analysis by SDS-destabilization and sucrose-density gradient centrifugation revealed that this complex is composed of at least two stable sub-complexes. The analysis further revealed that these two sub-complexes are likely to be connected by two IFT complex B proteins that either are present in both sub-complexes, or are excluded from both but act as a linker. These data suggest, that the IFT complex B is not, as previously described, acting as a single stable complex with proteins associated to the core structure. The biochemical analysis of the sub-complex structure shows, that there are two sub-modules that are closely linked. It remains unclear, if these sub-complexes exert one function as a tandem, or if they can act as separated modules within cilia or possibly within other microtubular structures.

The CoxD Protein, a Novel AAA+ ATPase Involved in Metal Cluster Assembly: Hydrolysis of Nucleotide-Triphosphates and Oligomerization

CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO2] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk), preparations of CoxD revealed a mean KM value of 0.69±0.14 mM ATP and an apparent Vmax value of 19.3±2.3 nmol ATP hydrolyzed min−1 mg−1. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer), preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10–16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO2] cluster assembly are discussed.

Involvement of lipid rafts in multiple signal transductions mediated by two isoforms of thromboxane A2 receptor: Dependency on receptor isoforms and downstream signaling types

Lipid rafts, microdomains in the plasma membrane, are known to be involved in G protein-coupled receptor signal transduction; however, their involvement in thromboxane A2 receptor (TP) signaling remains to be clarified. We examined whether two isoforms of TP, TPα and TPβ, utilize lipid rafts for multiple G protein signal transduction. Sucrose density gradient centrifugation followed by western blotting of HEK cells expressing TPα or TPβ revealed the localization of both TPα and TPβ in lipid rafts. Furthermore, methyl-β-cyclodextrin, which destroys lipid raft structure by depleting cholesterol, influenced G protein signaling elicited by TPα and TPβ to varying degrees. Phosphatidylinositol hydrolysis and cAMP accumulation induced by TPα or TPβ stimulation was markedly inhibited by methyl-β-cyclodextrin. In contrast, treatment with methyl-β-cyclodextrin partially inhibited RhoA activation induced by TPα stimulation, but failed to affect TPβ stimulation. Furthermore, the inhibitory action of methyl-β-cyclodextrin on cAMP accumulation was specific to TPα and TPβ, because methyl-β-cyclodextrin enhanced forskolin and β-adrenergic stimulation-induced cAMP accumulation. These results indicate that TP isoforms depend on lipid rafts during Gq and Gs signaling, while G13 signaling mediated by TP isoforms does not. Moreover, TPα seems to be more lipid raft-dependent with respect to RhoA activation than TPβ. These results indicate that the two isoforms of the TP mediate multiple signal transductions with varying degrees of lipid raft dependency. Moreover, our results provide a deeper understanding of the function of lipid rafts in G protein signaling and the physiological meaning of TP isoforms.

Translational regulation of Anopheles gambiae mRNAs in the midgut during Plasmodium falciparum infection

BackgroundMalaria is caused by Plasmodium parasites, which are transmitted via the bites of infected Anopheline mosquitoes. Midgut invasion is a major bottleneck for Plasmodium development inside the mosquito vectors. Malaria parasites in the midgut are surrounded by a hostile environment rich in digestive enzymes, while a rapidly responding immune system recognizes Plasmodium ookinetes and recruits killing factors from the midgut and surrounding tissues, dramatically reducing the population of invading ookinetes before they can successfully traverse the midgut epithelium. Understanding molecular details of the parasite-vector interactions requires precise measurement of nascent protein synthesis in the mosquito during Plasmodium infection. Current expression profiling primarily monitors alterations in steady-state levels of mRNA, but does not address the equally critical issue of whether the proteins encoded by the mRNAs are actually synthesized.ResultsIn this study, we used sucrose density gradient centrifugation to isolate actively translating Anopheles gambiae mRNAs based upon their association with polyribosomes (polysomes). The proportion of individual gene transcripts associated with polysomes, which is determined by RNA deep sequencing, reflects mRNA translational status. This approach led to identification of 1017 mosquito transcripts that were primarily regulated at the translational level after ingestion of Plasmodium falciparum-infected blood. Caspar, a negative regulator of the NF-kappaB transcription factor Rel2, appears to be substantially activated at the translational levels during Plasmodium infection. In addition, transcripts of Dcr1, Dcr2 and Drosha, which are involved in small RNA biosynthesis, exhibited enhanced associations with polysomes after P. falciparum challenge. This observation suggests that mosquito microRNAs may play an important role in reactions against Plasmodium invasion.ConclusionsWe analyzed both total cellular mRNAs and mRNAs that are associated with polysomes to simultaneously monitor transcriptomes and nascent protein synthesis in the mosquito. This approach provides more accurate information regarding the rate of protein synthesis, and identifies some mosquito factors that might have gone unrecognized because expression of these proteins is regulated mainly at the translational level rather than at the transcriptional level after mosquitoes ingest a Plasmodium-infected blood meal.

Three-dimensional structure of the Epstein–Barr virus capsid

Epstein-Barr virus (EBV), a gammaherpesvirus, infects >90 % of the world's population. Primary infection by EBV can lead to infectious mononucleosis, and EBV persistence is associated with several malignancies. Despite its importance for human health, little structural information is available on EBV. Here we report the purification of the EBV capsid by CsCl- or sucrose density-gradient centrifugation. Cryo-electron microscopy and image analysis resulted in two slightly different three-dimensional structures at about 20 Å resolution. These structures were compared with that of human herpesvirus 8, another gammaherpesvirus. CsCl-gradient purification leads to the removal of part of the triplex complex around the fivefold axes, whereas the complexes between hexons remained in place. This may be due to local differences in stability resulting from variation in quasi-equivalent interactions between pentons and hexons compared with those between hexons only.

Proteomic Identification of ADAM12 as a Regulator for TGF-β1-Induced Differentiation of Human Mesenchymal Stem Cells to Smooth Muscle Cells

BackgroundTransforming growth factor-β1 (TGF-β1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs.Methods and ResultsPretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-β1-induced differentiation of hASCs into smooth muscle cells.ConclusionsThese results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.

Development of monoclonal antibodies against bovine herpesvirus-1 (BHV-1)

The BHV-1 antigen was produced in bulk by propagating BHV-1 in Madin Darby bovine kidney cell line. The antigen purified by 20–40% sucrose density gradient centrifugation was confirmed for presence of virus. The BALB/C mice were immunized by purified virus via intraperitoneal route. The spleenic lymphocytes from immunized mice were fused with myeloma cells using polyethylene glycol as a fusion agent and suspended in HAT (hypoxanthine aminopterin thymidine) medium for differentiation of hybrids. A total of 11 clones were observed, out of which 7 were positive for antibody production. The supernatant of all the positive clones showed very low concentration of antibody, except the clone B6.1, where the concentration of antibody was higher than other positive clones. The culture supernatant of B6.1 hybrid clone was isotyped by enzyme linked immunosorbent assay (ELISA) based isotyping kit and isotype of monoclonal antibody was of IgM class. In western blotting, the IgM monoclonal antibody identified the 27 KD protein of BHV-1.

ADP-ribosylation Factor 1 Protein Regulates Trypsinogen Activation via Organellar Trafficking of Procathepsin B Protein and Autophagic Maturation in Acute Pancreatitis

Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.

Gold nanoparticle-biotinylated liposome hybrids as analytical reagents for biotin determination using a competitive assay and resonance light scattering detection

The preparation of hybrid nanostructures formed by gold nanoparticles (AuNPs) into biotinylated liposomes and their analytical application are presented. The surface of negatively charged AuNPs was modified with 1-dodecanethiol and the NPs were encapsulated into biotinylated liposomes using the rapid solvent evaporation method. Liposomes were resized by both mechanical shaking and ultrasound treatments and filled liposomes were separated from empty liposomes using sucrose density gradient centrifugation. The analytical usefulness of AuNP-liposome hybrids as amplification probes for biotin determination was checked using the competitive affinity reaction based on the avidin–biotin interaction and biotilynated phospholipids for the synthesis of the liposome hybrids. The method was automatized using a flow system and measuring the resonance light scattering signal. The dynamic range of the calibration graph was 0.001–20μgmL−1, (r2=0.9998, n=14), with a detection limit of 0.3ngmL−1. The precision, expressed as relative standard deviation (RSD%), was lower than 5% and the sampling frequency was 9h−1. The approach has been applied to the determination of biotin in food samples, with recovery values ranging between 88.2 and 105.2%.

A dynamic plasma membrane proteome analysis of alcohol-induced liver cirrhosis

Alcohol-induced injury has become one of the major causes for liver cirrhosis. However, the molecular mechanisms of ethanol-induced injury are not fully understood. To this end, we performed a dynamic plasma membrane proteomic research on rat model. A rat model from hepatitis to liver cirrhosis was developed. Plasma membrane from liver tissue with liver fibrosis stage of 2 and 4 (S2 and S4) was purified by sucrose density gradient centrifugation. Its purification was verified by western blotting. Proteins from plasma membrane were separated by two-dimensional electrophoresis (2DE) and differentially expressed proteins were identified by tandem mass spectrometry. 16 consistent differentially expressed proteins from S2 to S4 were identified by mass spectrometry. The expression of differentially expressed proteins annexin A6 and annexin A3 were verified by western blotting, and annexin A3 was futher verified by immunohistochemistry. Our research suggests a possible mechanism by which ethanol alters protein expression to enhance the liver fibrosis progression. These differentially expressed proteins might be new drug targets for treating alcoholic liver cirrhosis.

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments

Disabled-2 (Dab2) inhibits Wnt/β-catenin signalling by binding LRP6 and promoting its internalization through clathrin

Canonical Wnt signalling requires caveolin-dependent internalization of low-density lipoprotein receptor-related protein 6 (LRP6). Here we report that the tumour suppressor and endocytic adaptor disabled-2 (Dab2), previously described as an inhibitor of Wnt/β-catenin signalling, selectively recruits LRP6 to the clathrin-dependent endocytic route, thereby sequestering it from caveolin-mediated endocytosis. Wnt stimulation induces the casein kinase 2 (CK2)-dependent phosphorylation of LRP6 at S1579, promoting its binding to Dab2 and internalization with clathrin. LRP6 receptor mutant (S1579A), deficient in CK2-mediated phosphorylation and Dab2 binding, fails to associate with clathrin, and thus escapes the inhibitory effects of Dab2 on Wnt/β-catenin signalling. Our data suggest that the S1579 site of LRP6 is a negative regulatory point during LRP6-mediated dorsoventral patterning in zebrafish and in allograft mouse tumour models. We conclude that the tumour suppressor functions of Dab2 involve modulation of canonical Wnt signalling by regulating the endocytic fate of the LRP6 receptor.

Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp0 mutant of Bacillus subtilis triggers YvyD‐dependent dimerization of ribosome

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-β-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the ΔrelA ΔyjbM ΔywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.

Subunit Interactions and Organization of the Chlamydomonas reinhardtii Intraflagellar Transport Complex A Proteins

Chlamydomonas reinhardtii intraflagellar transport (IFT) particles can be biochemically resolved into two smaller assemblies, complexes A and B, that contain up to six and 15 protein subunits, respectively. We provide here the proteomic and immunological analyses that verify the identity of all six Chlamydomonas A proteins. Using sucrose density gradient centrifugation and antibody pulldowns, we show that all six A subunits are associated in a 16 S complex in both the cell bodies and flagella. A significant fraction of the cell body IFT43, however, exhibits a much slower sedimentation of ∼2 S and is not associated with the IFT A complex. To identify interactions between the six A proteins, we combined exhaustive yeast-based two-hybrid analysis, heterologous recombinant protein expression in Escherichia coli, and analysis of the newly identified complex A mutants, ift121 and ift122. We show that IFT121 and IFT43 interact directly and provide evidence for additional interactions between IFT121 and IFT139, IFT121 and IFT122, IFT140 and IFT122, and IFT140 and IFT144. The mutant analysis further allows us to propose that a subset of complex A proteins, IFT144/140/122, can form a stable 12 S subcomplex that we refer to as the IFT A core. Based on these results, we propose a model for the spatial arrangement of the six IFT A components.

Effects of noble gas conditioning on Caveolin expression in the rat heart in vivo

Caveolins are structural scaffolding proteins that allow for organization of signalling molecules in caveolae, membrane invaginations enriched in lipids. Caveolin‐3 (Cav‐3) is a heart specific isoform that is modulated by volatile anesthetics and is required for cardiac protection. We hypothesized that the cardioprotective noble gases Helium (He) and Xenon (Xe) also influence the enrichment of Cav‐3 to caveolae in vivo. Sprague‐ Dawley rats underwent 25 minutes of myocardial ischemia and 2 hours of reperfusion. Rats where left untreated (Con) or underwent different preconditioning (PC) protocols: in He late PC (HeLPC), rats inhaled He for 3×5 min a day before the experiment, the He postconditioning (HePost) group inhaled 70% He for 15 min at the onset of reperfusion. The Xe‐PC rats inhaled 70% Xe for 3×5 min before ischemia. Hearts were processed for sucrose density gradient centrifugation to purify caveolae. Cav‐3 and 1 expression were determined by immunoblotting. HeLPC and HePost increased Cav‐3 localization from 0.2±0.1 in Con to 0.4±0.1 and 0.4±0.2 (arbitrary units), respectively, in buoyant caveolar fractions indicative of increased caveolae formation. Xe‐PC had no effect on Cav‐3 localization. We found no effect on Cav‐1 localization.These data suggest that He might exert its cardioprotective effect by augmenting Cav‐3 localization to caveolae to further enhance cardioprotective signaling.

Ribosome Fractionation in Yeast

This protocol describes yeast ribosome fractionation in the gradient of sucrose. During the cyclic process of translation, a small (40S) and large (60S) ribosomal subunit associate with mRNA to form an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation, and then dissociates into the 40S and 60S subunits on termination. During elongation by one ribosome, further ribosomes can initiate translation on the same mRNA to form polysomes. The mass of each polysomal complex is determined primarily by the number of ribosomes it contains. Hence, the population of polysomes within the cell can be size-fractionated by sucrose density gradient centrifugation on the basis of the loading of ribosomes on the mRNA. Several compounds help to maintain or to disrupt the polysomes (Figure 1).

Polysome Preparation, RNA Isolation and Analysis.

During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosome can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.