Differential Centrifugation Procedure Research Articles

323 Advances in using membrane vesicles for studying intestinal functions

Abstract Enterocytes are the major cell lineage of the small intestinal epithelia along the crypt-villus axis in gut mucosa. Enterocytes participate in the gut terminal nutrient digestion and absorption; and have essential roles in nutrient and metabolite recycling and efflux, nutrient and hormonal sensing, interactions with symbiotic microbiome and binding to infectious viruses, thus influencing the host homeostasis and whole-body physiological status. Intestinal membrane vesicles have been widely used for studying various gut functions in the past several decades. Villus enterocytic brush border membrane and the apical membrane vesicles along the crypt-villus axis in gut mucosa can be prepared and isolated by Mg2+- and Ca2+-precipitation and differential centrifugation procedures. Whereas, gut mucosal basolateral membrane vesicles can be prepared, fractionated and differentiated from other intracellular organelles such as the mitochondrial inner membrane by the Percoll gradient ultracentrifugation. Nutrient transport and exchange activities in the membrane vesicles in vitro are conducted often by rapid hand filtration, and in excess, in an automated fast sampling apparatus. Nutrient transport and exchange activity kinetics are derived according to the well-established classic Michaelis-Menten and the tracer inhibitory kinetic models with a simple linear diffusional component. Physiological and biochemical properties associated with various nutrient uptake systems such as pH optimum, requirements for co-transported ions (e.g., Na+, K+, Cl-), transporter affinity and substrate specificity can be effectively investigated in vitro with the membrane vesicles by altering the compositions of uptake buffers and intra-vesicular pre-loading buffers. Some of the fundamental gut functions are established via using the membrane vesicles in different mammalian species. And these include H+-peptide cotransport, Na+-cotransport and Na+-independent systems of hexose uptake across the apical membrane and the low-affinity Na+-independent hexose bidirectional uptake across the basolateral membrane that were characterized in rodents; Na+-dependent cotransport and Na+-independent systems of amino acid (AA) transport across the apical membrane that were revealed in rodents and pigs; as well as the Na+-hexose and AA cotransport and the co-expression of the coronavirus SARS-CoV-2 host epithelial apical membrane binding receptor, i.e., the angiotensin-converting enzyme-2 (ACE2), along the gut crypt-villus axis that were identified in liquid formula-fed piglets. In the post genomic and metagenomic sequencing era, further research efforts need to pursue understanding of expression and various functionality of the gut terminal hydrolases, nutrient transporters, nutrient exchangers, nutrient sensors, substrate and particle receptors, taste receptors, viral binding receptors such as ACE2 and hormonal receptors and their interactions with luminal factors such as nutrient, metabolites, symbiotic bacterial outer membrane vesicles etc., along the longitudinal and crypt-villus axes, through using the mammalian primary enterocytic apical and the basolateral membrane vesicles under various research settings.

PSII-13 Size and Abundance of Extracellular Vesicles in Cattle During Early to Mid-Gestation Consuming Control Or Restricted Diets

Abstract Extracellular vesicles (EVs) are known to be present in cattle blood, and have been shown to contain miRNA; however, little information is available describing the abundance or size of circulating EVs in cattle during gestion. Our objective was to determine EVs size and abundance in maternal circulation during early and mid-gestation. Cattle were blocked by body condition score (BCS) and body weight (BW), synchronized and bred to the same AI sire using male-sexed semen (n = 23). At day 30 post-AI, pregnancy was determined and cattle were allotted to 1 of 3 groups and received a control diet (CON) from d 30 to d 190 of gestation (n = 8), a CON diet from d 30 to d 110 and nutrient-restricted (NR) diet from d 110 to d 190 (CON/NR; n = 8), or an NR diet from d 30 to d 110 and CON diet from d 110 to d 190 (NR/CON; n = 7). BW and BCS were recorded weekly or biweekly, respectively. Blood samples were collected via the coccygeal vein, samples allowed to clot, stored at 4 oC overnight and processed for serum. Serum was stored at -20 oC until used for EVs isolation. Six serum samples were utilized for each diet within each day of gestation. Two samples within each group were pooled giving 3 biological replicates for each group by day combination. EVs were isolated using differential centrifugation procedures, resuspended in 1X PBS and then subjected to nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Panalytical, UK). NTA established a size range across all samples of 31 to over 400 nm indicating the presence of exosomes and larger EVs. NTA data were subjected to ANOVA where day of gestation, diet treatment, and their interaction were the main effects. Student’s t-test among LS means for particle EVs concentration and size were used to determine significance (P < 0.05). Day of gestation affected EVs concentration with 3.9 x 109 ± 2.1 x 108 particles per ml, 1.4 x 109 ± 2.1 x 108 particles per mL and 2.6 x 109 ± 2.2 x 108 particles per mL, at day 30, 110 and 190 of gestation, respectively (P < 0.05). There was a day of gestation by diet effect for the size of EVs with the largest sizes observed for CON, NR and CON/NR group at day 30 and for NR/CON at day 190 (P < 0.05). The smallest EVs were detected on day 110 of gestation for the NR/CON and the CON groups (P < 0.05). These data indicate that EVs size in maternal circulation can be impacted by diet and day of gestation and this observation is most likely linked to the observations of differential abundance of miRNA previously reported.

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures

IntroductionThis study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. MethodsFifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. ResultsA total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. ConclusionThis method provides rapid bacterial identification and AST, offering definitive results 24–48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures

IntroductionThis study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. MethodsFifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. ResultsA total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. ConclusionThis method provides rapid bacterial identification and AST, offering definitive results 24–48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.

Scaled preparation of extracellular vesicles from conditioned media.

Extracellular vesicles (EVs) especially of mesenchymal stem/stomal cells (MSCs) are increasingly considered as biotherapeutic agents for a variety of different diseases. For translating them effectively into the clinics, scalable production processes fulfilling good manufacturing practice (GMP) are needed. Like for other biotherapeutic agents, the manufacturing of EV products can be subdivided in the upstream and downstream processing and the subsequent quality control, each of them containing several unit operations. During upstream processing (USP), cells are isolated, stored (cell banking) and expanded; furthermore, EV-containing conditioned media are produced. During downstream processing (DSP), conditioned media (CM) are processed to obtain concentrated and purified EV products. CM are either stored until DSP or are directly processed. As first unit operation in DSP, clarification removes remaining cells, debris and other larger impurities. The key operations of each EV DSP is volume-reduction combined with purification of the concentrated EVs. Most of the EV preparation methods used in conventional research labs including differential centrifugation procedures are limited in their scalability. Consequently, it is a major challenge in the therapeutic EV field to identify appropriate EV concentration and purification methods allowing scale up. As EVs share several features with enveloped viruses, that are used for more than two decades in the clinics now, several principles can be adopted to EV manufacturing. Here, we introduce and discuss volume reducing and purification methods frequently used for viruses and analyze their value for the manufacturing of EV-based therapeutics.

Subcellular partitioning profiles and metallothionein levels in indigenous clams Moerella iridescens from a metal-impacted coastal bay

In this study, the effect of environmental metal exposure on the accumulation and subcellular distribution of metals in the digestive gland of clams with special emphasis on metallothioneins (MTs) was investigated. Specimens of indigenous Moerella iridescens were collected from different natural habitats in Maluan Bay (China), characterized by varying levels of metal contamination. The digestive glands were excised, homogenized and six subcellular fractions were separated by differential centrifugation procedures and analyzed for their Cu, Zn, Cd and Pb contents. MTs were quantified independently by spectrophotometric measurements of thiols. Site-specific differences were observed in total metal concentrations in the tissues, correlating well with variable environmental metal concentrations and reflecting the gradient trends in metal contamination. Concentrations of the non-essential Cd and Pb were more responsive to environmental exposure gradients than were tissue concentrations of the essential metals, Cu and Zn. Subcellular partitioning profiles for Cu, Zn and Cd were relatively similar, with the heat-stable protein (HSP) fraction as the dominant metal-binding compartment, whereas for Pb this fraction was much less important. The variations in proportions and concentrations of metals in this fraction along with the metal bioaccumulation gradients suggested that the induced MTs play an important role in metal homeostasis and detoxification for M. iridescens in the metal-contaminated bay. Nevertheless, progressive accumulation of non-essential metals (Cd, and especially Pb) resulting from “spillover” was observed in putative metal- sensitive (e.g., mitochondria and heat-denaturable protein (HDP)) or lysosome/microsome fractions, demonstrating that metal detoxification was incomplete and increased the toxicological risk to M. iridescens inhabiting the metal-impacted environments. Through multiple stepwise regression analysis, the induction of MTs was statistically correlated with the HSP concentrations of Cu, and to a lesser extent with Zn, and ultimately to the Cd concentrations, exhibiting significant dose-dependent relationships. Overall, these findings not only revealed the fates of accumulated metals, but scientifically favored an improved understanding of the detoxification at the subcellular level in response to metal accumulation, supporting the focus of metabolic availability assessment on the intracellular processes or events occurring within organisms.

Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains.

BackgroundOuter membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.Methodology/Principal FindingsOMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.Conclusion/SignificanceBiological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.

Detection of soluble BAG3 and anti-BAG3 antibodies in patients with chronic heart failure

Dear Editor, Bcl-2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones that interact with the ATPase domain of the heat shock protein HSP70 through the BAG domain (amino acids 110–124). BAG3 contains also a WW domain, a proline-rich repeat (PXXP) and two conserved Ile-Pro-Val motifs that mediate binding to other proteins.1 bag3 gene is constitutively expressed in a few normal cell types, including myocytes, and in several primary tumors, while its expression is inducible by stressors in other cell types. Many evidence indicate that BAG3 has a role in sustaining cell survival, through mechanisms that, depending on the cell context, rely on the ability of BAG3 to modulate levels or localization of apoptosis-regulating proteins, such as IKKγ, Bax or BRAF, in either an Hsp70-dependent or -independent fashion.1, 2, 3 BAG3 is expressed during cardiomyoblast differentiation and sustains myogenin expression. In cardiomyocytes, BAG3 protein localizes at the Z-disc and interacts with the actin capping protein, CapZβ1, stabilizing myofibril structure; BAG3 mutations can impair the Z-disc assembly and increase the sensitivity to stress-induced apoptosis. Mutations in bag3 gene have been associated with some forms of myofibrillar myopathy and dilated cardiomyopathy.4, 5, 6, 7 Interestingly, we detected BAG3 protein in supernatants of the rat cardiomyocyte cell line H9c2 and adult Human Cardiac Myocytes (HCMa) after 16 h of serum deprivation (Figure 1a). We then verified that BAG3 was present in extracellular vesicles isolated through a differential centrifugation procedure (Supplementary Figure S1A). To investigate the possibility that BAG3 could be released in vivo and become detectable in sera in conditions in which the heart is undergoing chronic stress, we analyzed sera from two patients affected by chronic heart failure (CHF). By western blot analysis, we detected a band recognized by anti-BAG3 antibody at the expected molecular weight in sera from patients but not from healthy donors (Figure 1b). We excised the band from a replica gel and subjected it to mass spectrometry, unmistakably identifying BAG3 (Supplementary Figure S1B). Furthermore, we found that sera from CHF patients recognized BAG3 protein in western blotting, using an anti-human IgG as secondary antibody (Supplementary Figure S1C). This result indicated the presence of anti-BAG3 antibodies in CHF patients' sera. To confirm this finding, we developed an ELISA test using recombinant BAG3 to coat plates and anti-human IgG to reveal and analyzed sera from 52 CHF patients (EF<45%), compared with sera from 84 healthy donors. As shown in Figure 1c (and in Supplementary Figure S1D), we detected significantly higher values of anti-BAG3 antibodies in patients' compared with controls' sera. These data suggest that upon cardiac stress cardiomyocytes release BAG3 and this in turn results in production of auto-antibodies. There is no correlation with NYHA scores and antibody levels at this stage but screening of a larger number of patients in the future might be necessary to reveal potential correlations. Figure 1 (a) Detection of BAG3 protein in supernatants from cultured cardiomyocytes. Human (HCMa) and rat (H9c2) cardiomyocytes, at 80% confluence, were incubated with or without 10% FBS for 16 h at 37 °C in a 5% ... These results describe for the first time an extracellular BAG3 (eBAG3) released by stressed cardiomyocytes. As BAG3 lacks the consensus signal required for secretion via ER–Golgi pathway, it is likely to be released by the non-classical secretory pathway.8 eBAG3 release by stressed cardiomyocytes appears to result in production of auto-antibodies that could potentially be used as a biomarker for CHF patients, in combination with other already established markers. The presence of anti-BAG3 antibodies in CHF patients' sera indicates that released BAG3 can activate the immune system, and might therefore exert positive or negative functional effects on cardiac function, depending on the context. Future studies are required to clarify the biological roles of BAG3 and anti-BAG3 antibodies in CHF, and the utility of anti-BAG3 antibodies as a tool contributing to the study of the disease.

Subcellular differences in handling Cu excess in three freshwater fish species contributes greatly to their differences in sensitivity to Cu

Since changes in metal distribution among tissues and subcellular fractions can provide insights in metal toxicity and tolerance, we investigated this partitioning of Cu in gill and liver tissue of rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and gibel carp (Carassius auratus gibelio). These fish species are known to differ in their sensitivity to Cu exposure with gibel carp being the most tolerant and rainbow trout the most sensitive. After an exposure to 50μg/l (0.79μM) Cu for 24h, 3 days, 1 week and 1 month, gills and liver of control and exposed fish were submitted to a differential centrifugation procedure. Interestingly, there was a difference in accumulated Cu in the three fish species, even in control fishes. Where the liver of rainbow trout showed extremely high Cu concentrations under control conditions, the amount of Cu accumulated in their gills was much less than in common and gibel carp. At the subcellular level, the gills of rainbow trout appeared to distribute the additional Cu exclusively in the biologically active metal pool (BAM; contains heat-denaturable fraction and organelle fraction). A similar response could be seen in gill tissue of common carp, although the percentage of Cu in the BAM of common carp was lower compared to rainbow trout. Gill tissue of gibel carp accumulated more Cu in the biologically inactive metal pool (BIM compared to BAM; contains heat-stable fraction and metal-rich granule fraction). The liver of rainbow trout seemed much more adequate in handling the excess Cu (compared to its gills), since the storage of Cu in the BIM increased. Furthermore, the high % of Cu in the metal-rich granule fraction and heat-stable fraction in the liver of common carp and especially gibel carp together with the better Cu handling in gill tissue, pointed out the ability of the carp species to minimize the disadvantages related to Cu stress. The differences in Cu distribution at the subcellular level of gills and liver of these fish species strongly reflects their capacity to handle Cu excess and is one of the greatest contributors to their difference in sensitivity to Cu.

Effect of Transient and Permanent Permeability Transition Pore Opening on NAD(P)H Localization in Intact Cells

To study the effect of mitochondrial permeability transition pore (PTP) opening on NAD(P)H localization, intact cells were exposed to the Ca(2+) ionophore A23187. PTP opening, mitochondrial membrane potential, mitochondrial volume, and NAD(P)H localization were assessed by time-lapse laser confocal microscopy using the calcein-cobalt technique, tetramethylrhodamine methyl ester, MitoTracker, and NAD(P)H autofluorescence, respectively. Concomitant with PTP opening, NAD(P)H fluorescence increased outside mitochondria. These events occurred in all cells and were prevented by cyclosporin A. Mitochondrial membrane potential was not systematically collapsed, whereas mitochondrial volume did not change, confirming that A23187 induced transient PTP opening in a subpopulation of cells and suggesting that mitochondrial swelling did not immediately occur after PTP opening in intact cells. NAD(P)H autofluorescence remained elevated after PTP opening, particularly after membrane potential had been collapsed by an uncoupler. Extraction of nucleotide for NAD(P)H quantification confirmed that PTP opening led to an increase in NAD(P)H content. Because the oxygen consumption rate decreased, whereas the lactate/pyruvate ratio increased after PTP opening in intact cells, we conclude that PTP opening inhibits respiration and dramatically affects the cytosolic redox potential in intact cells.

Recent advances using stem cell‐derived cardiac and vascular cells for cardiomyoplasty

Stem cells of either adult or embryonic origin (embryonic stem cells, ES cells) are an unlimited source of cells that could be used for cell transplantation approaches to treat cardiac infarction and to overcome the shortage of donor organs. Whereas adult stem cell transplantation has already reached the clinical state, the research with ES cells is still in its infancy which is related to the danger of teratoma formation within the cell recipient as well as due to potential problems arising from immune responses towards the transplanted cells. Although some improvement in cardiac function has been observed so far using adult stem cell transplantation, the so far achieved benefit is not sufficient to allow routine patient treatment. Furthermore, the transdifferentiation of adult stem cells into cardiac cells that was reported in several studies could not be reproduced by other groups, and independent researchers reported that transdifferentiation was indeed a misinterpretation resulting from fusion of stem cells with adjacent cardiac cells. In contrast to adult stem cells the cardiovascular differentiation potential of ES cells is undoubted, and due to the huge proliferation capacity of ES cells this stem cell species may represent the most promising stem cell population that may be applied in future clinical approaches. However, besides problems of immune rejection which could be overcome with conventional immunosuppressants these cells bear the danger of teratoma formation which is arising from transplantation of still undifferentiated cells into the host organ. Several approaches have been elaborated during recent years to cope with teratoma generation. The most straightforward procedures to remove undifferentiated ES cells from preparations to be transplanted into organs are: antibiotic selection procedures, fluorescence‐activated cell sorting, differential centrifugation procedures, and cytotoxic antibodies directed against the undifferentiated cell population. One of the latest developments is the derivation of so called “induced ES cells”. These cells are gained by reprogramming somatic cells through introduction of stemness genes like Oct4, Sox2, Klf4, and c‐Myc. Upon transfection with these genes somatic cells achieve an ES cell phenotype and can be differentiated into cells of all three germ layers. Using induced ES cells immunological problems arising from allogeneic stem cell transplantation could be overcome since these stem cells are produced from the patients′ somatic cells thus carrying his immunological features. Besides scientific progress with the “classical” adult and ES cells, research of recent years came up with resident cardiac stem cells that reside in stem cell niches within the adult heart. It has been suggested that resident stem cells may be activated in situations of cardiac injury and may represent an intrinsic mechanism of cardiac repair, thus overcoming the previously generally accepted concept of the heart as a post‐mitotic organ. Currently researchers are trying to propagate resident cardiac stem cells in cell culture to scale up cell numbers for cell transplantation approaches. The transplantation procedures of stem cells are assisted by rapidly evolving tissue engineering procedures which utilize biodegradable scaffolds to fix transplanted cells within the transplantation site. Recent research has shown that successful engraftment of transplanted stem cell tissues into the infarcted heart can only be achieved if not only cardiomyocytes are transplanted into the area of infarction but additionally endothelial cells, smooth muscle cells and fibroblast ‐ which in concert built up the vascularized microenvironment that is necessary for cardiac cell proliferation and cell survival.

Solubilization of Protein Aggregates by the Acid Stress Chaperones HdeA and HdeB

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

Dimeric Structure of Human Na+/H+ Exchanger Isoform 1 Overproduced in Saccharomyces cerevisiae

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% alpha-helix, 23% beta-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1.

Human Mitochondrial Ribosomal Protein MRPL12 Interacts Directly with Mitochondrial RNA Polymerase to Modulate Mitochondrial Gene Expression

The core human mitochondrial transcription machinery comprises a single subunit bacteriophage-related RNA polymerase, POLRMT, the high mobility group box DNA-binding protein h-mtTFA/TFAM, and two transcriptional co-activator proteins, h-mtTFB1 and h-mtTFB2 that also have rRNA methyltransferase activity. Recapitulation of specific initiation of transcription in vitro can be achieved by a complex of POL-RMT, h-mtTFA, and either h-mtTFB1 or h-mtTFB2. However, the nature of mitochondrial transcription complexes in vivo and the potential involvement of additional proteins in the transcription process in human mitochondria have not been extensively investigated. In Saccharomyces cerevisiae, transcription and translation are physically coupled via the formation of a multiprotein complex nucleated by the binding of Nam1p to the amino-terminal domain of mtRNA polymerase (Rpo41p). This model system paradigm led us to search for proteins that interact with POLRMT to regulate mitochondrial gene expression in humans. Using an affinity capture strategy to identify POL-RMT-binding proteins, we identified mitochondrial ribosomal protein L7/L12 (MRPL12) as a protein in HeLa mitochondrial extracts that interacts specifically with POLRMT in vitro. Purified recombinant MRPL12 binds to POLRMT and stimulates mitochondrial transcription activity in vitro, demonstrating that this interaction is both direct and functional. Finally, from HeLa cells that overexpress FLAG epitope-tagged MRPL12, increased steady-state levels of mtDNA-encoded transcripts are observed and MRPL12-POLRMT complexes can be co-immunoprecipitated, providing strong evidence that this interaction enhances mitochondrial transcription or RNA stability in vivo. We speculate that the MRPL12 interaction with POLRMT is likely part of a novel regulatory mechanism that coordinates mitochondrial transcription with translation and/or ribosome biogenesis during human mitochondrial gene expression.

Bidirectional Replication Initiates at Sites Throughout the Mitochondrial Genome of Birds

Analysis of mitochondrial replication intermediates of Gallus gallus on fork-direction gels indicates that replication occurs in both directions around circular mitochondrial DNA. This finding was corroborated by a study of chick mitochondrial DNA on standard neutral two-dimensional agarose gels, which yielded archetypal initiation arcs in fragments covering the entire genome. There was, however, considerable variation in initiation arc intensity. The majority of initiation events map to regions flanking the major non-coding region, in particular the NADH dehydrogenase subunit 6 (ND6) gene. Initiation point mapping of the ND6 gene identified prominent free 5' ends of DNA, which are candidate start sites for DNA synthesis. Therefore we propose that the initiation zone of G. gallus mitochondrial DNA encompasses most, if not all, of the genome, with preferred initiation sites in regions flanking the major non-coding region. Comparison with mammals suggests a common mechanism of initiation of mitochondrial DNA replication in higher vertebrates.

Sub-cellular partitioning of metals (Cd, Cu, Zn) in the gills of a freshwater bivalve, Pyganodon grandis: role of calcium concretions in metal sequestration

Indigenous unionid molluscs, Pyganodon grandis, were collected from nine lakes in the Rouyn–Noranda area (Québec, Canada) along a polymetallic concentration gradient (Cd, Cu, Zn). After excision, the gills were gently homogenised and the cellular compartments were separated by a differential centrifugation procedure that yielded the following particulate fractions: “nuclei + cellular debris”, “mitochondria”, “lysosomes + microsomes” and “granules”. The supernatant remaining after the final ultracentrifugation step, i.e., the operationally-defined cytosol, was separated into a “heat-denaturable proteins” (HDP) fraction and a “heat-stable proteins” (HSP) fraction containing metallothionein (MT). The Cd, Cu and Zn content of each particulate and cytosolic fraction was determined and gill metallothionein was quantified independently by a mercury saturation assay. Cytosolic Cd concentrations were significantly related to the dissolved Cd concentrations at each site, but cytosolic Cu and Zn (essential metals) were not related to their respective ambient dissolved metal concentrations. Metallothionein concentrations increased along the metal contamination gradient and were related to cytosolic Cd (and Zn) in a concentration-dependent manner. However mass balance calculations showed that binding to metallothionein could only account for a small proportion of total gill metal (∼10% Cd; ∼3% Cu; ∼1% Zn). Under these chronic exposure conditions, the three metals (Cd, Cu and Zn) were mainly located in calcium concretions present in the gills (respectively 58 ± 13% of the total gill Cd, 64 ± 6% of the total gill Cu and 73 ± 6% of the total gill Zn). The overall contribution of granules to the total gill dry weight remained relatively constant among the different lakes, suggesting that lake-to-lake variations in granule synthesis were independent of the metal contamination gradient, i.e., these constituent elements of unionid gills act as non-inducible metal sinks at the cellular level. Metal concentrations increased proportionally in both the granules and the MT pool along the polymetallic gradient, suggesting a constant partitioning between these two compartments. Overall, despite an increase in Cd in the “mitochondria” fraction, metal sequestration mechanisms seem to be reasonably effective in detoxifying cadmium: in the cytosol, Cd concentrations in the potentially metal-sensitive HDP fraction remained relatively low and constant, even in specimens collected from the most contaminated lakes.

Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes.

The legume Rhizobium symbiosis leads to the formation of a new compartment in the plant cell, the symbiosome. This compartment harbours the bacteroids surrounded by a peribacteroid membrane (PBM) originating from the plant plasma membrane. The PBM and the space between the PBM and the bacteroid membrane, called peribacteroid space (PS), mediate the exchange of metabolites between the symbionts. Proteome analysis was used as an approach to characterise the proteins in the PBM and the PS. A standard differential centrifugation procedure including a Percoll gradient was used for symbiosome isolation from pea root nodules. Proteins in the PBM and PS fractions obtained from the symbiosomes were separated by two-dimensional gel electrophoresis, and 89 spots were analysed by tandem mass spectrometry. The proteins of 46 spots could be identified by database search. The results showed that PS and even PBM preparations from pea symbiosomes always contain abundant amounts of bacteroid proteins as a contaminate. Interestingly, in addition to a few PS/PBM proteins a number of endomembrane proteins (less likely representing a contaminate), including V-ATPase, BIP, and an integral membrane protein known from COPI-coated vesicles, were found in the PBM fraction, supporting the role of the endomembrane system in PBM biogenesis.

Isolation and characterization of actinomycetes from Brazilian tropical soils

Actinomycetes have been isolated from three Brazilian tropical soils. The dispersion and differential centrifugation procedure revealed count values 1.5 to 5.0 times greater than those obtained by the conventional dilution plate technique for all soils and media tested. Eighteen strains, promising for biotechnological applications, were submitted to chemotaxonomic procedures and numerical taxonomy for identification. Two were identified as Amycolatopsis orientalis, one as Streptomyces misakiensis, and two tentatively included or associated to S. chromofuscus and S. griseoruber. The others, all belonging to the Streptomyces genus, could not be fitted into any known species, and were arranged by the UPGMA analysis for classification, as an isolated group. This suggests that the actinomycetes in tropical soils may represent a vast unexplored resource for biotechnology.

Isolation of Subcellular Fractions from the YeastSaccharomyces cerevisiae

This unit presents detailed protocols for a range of centrifugation-based subcellular fractionation procedures for the yeast Saccharomyces cerevisiae. Techniques include spheroplast preparation, glass-bead lysis, differential centrifugation, and several density gradient procedures using a variety of gradient media. There are analytical procedures that are primarily designed to evaluate the association of proteins with organelles in the exocytic and endocytic pathways. Additionally, there are preparative protocols for isolation of yeast nuclei, vacuoles, mitochondria, peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. The unit also contains a table, with references, for alternative approaches to isolation of these organelles and fractions.

Efficiency of the dispersion and differential centrifugation technique in the isolation of chitinolytic actinomycetes from soil

Actinomycetes were isolated from an acidic Brazilian soil under cerrado (savanna) vegetation, previously amended with chitin. The efficiency of two isolation techniques, the conventional dilution plate and the dispersion and differential centrifugation (DDC) procedures, were compared and a mathematical computer analysis of the results was made. The DDC technique gave counts about nine times greater than the conventional one. Analysis of the derivative curves suggested the presence of two distinct groups of actinomycetes on the isolation plates, one able to induce chitinolytic enzymes during amendment and the other whose initial activity was delayed. Major differences in results with different isolation procedures suggest a more subtle ecological role for these microbes in terms of their location in the soil environment.