Change In Buoyant Density Research Articles

Microaerobic methane-driven denitrification in a biotrickle bed – Investigating the active microbial biofilm community composition using RNA-stable isotope probing

A microaerobic (2% O2 v/v) biotrickle bed reactor supplied continuously with 2% methane to drive nitrate removal (MAME-D) was investigated using 16S rDNA and rRNA amplicon sequencing in combination with RNA-stable isotope probing (RNA-SIP) to identify the active microorganisms. Methane removal rates varied from 500 to 1000 mmol m−3h−1 and nitrate removal rates from 25 to 58 mmol m−3h−1 over 55 days of operation. Biofilm samples from the column were incubated in serum bottles supplemented with 13CH4. 16S rDNA analysis indicated a simple community structure in which four taxa accounted for 45% of the total relative abundance (RA). Dominant genera included the methanotroph Methylosinus and known denitrifiers Nubsella and Pseudoxanthomonas; along with a probable denitrifier assigned to the order Obscuribacterales. The 16S rRNA results revealed the methanotrophs Methylocystis (15% RA) and Methylosinus (10% RA) and the denitrifiers Arenimonas (10% RA) and Pseudoxanthomonas (7% RA) were the most active genera. Obscuribacterales was the most active taxa in the community at 22% RA. Activity was confirmed by the Δ buoyant density changes with time for the taxa, indicating most of the community activity was associated with methane oxidation and subsequent consumption of methanotrophic metabolic intermediates by the denitrifiers. This is the first report of RNA stable isotope probing within a microaerobic methane driven denitrification system and the active community was markedly different from the full community identified via 16S-rDNA analysis.

Golgi-associated Maturation of Very Low Density Lipoproteins Involves Conformational Changes in Apolipoprotein B, but Is Not Dependent on Apolipoprotein E

The major protein component in secreted very low density lipoproteins (VLDL) is apoB, and it is established that these particles can reach sizes approaching 100 nm. We previously employed a cell-free system to investigate the nature of the vesicles in which this large cargo exits the endoplasmic reticulum (ER) (Gusarova, V., Brodsky, J. L., and Fisher, E. A. (2003) J. Biol. Chem. 278, 48051-48058). We found that apoB-containing lipoproteins exit the ER as dense lipid-protein complexes regardless of the final sizes of the particles and that further expansion occurs via post-ER lipidation. Here, we focused on maturation in the Golgi apparatus. In three separate approaches, we found that VLDL maturation (as assessed by changes in buoyant density) was associated with conformational changes in apoB. In addition, as the size of VLDL expanded, apoE concentrated in a subclass of Golgi microsomes or Golgi-derived vesicles that co-migrated with apoB-containing microsomes or vesicles, respectively. A relationship between apoB and apoE was further confirmed in co-localization studies by immunoelectron microscopy. These combined results are consistent with previous suggestions that apoE is required for VLDL maturation. To our surprise, however, we observed robust secretion of mature VLDL when apoE synthesis was inhibited in either rat hepatoma cells or apoE(-/-) mouse primary hepatocytes. We conclude that VLDL maturation in the Golgi involves apoB conformational changes and that the expansion of the lipoprotein does not require apoE; rather, the increase in VLDL surface area favors apoE binding.

Activation of the tumor suppressor merlin modulates its interaction with lipid rafts.

Neurofibromatosis type 2 (NF2) is a genetic disorder characterized by bilateral schwannomas of the eighth cranial nerve. The NF2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane/F-actin linkers. Merlin resists solubilization by the detergent Triton X-100 (TX-100), a property commonly attributed to association with the cytoskeleton. Accordingly, NF2 patient mutations that encode merlins with enhanced TX-100 solubility have been explained previously in terms of loss of cytoskeletal attachment. However, here we present data to suggest that the detergent resistance of merlin is a result of its constitutive residence in lipid rafts. Furthermore, when cells are grown to high density, merlin shifts to a more buoyant lipid raft fraction in a density gradient. This shift is mimicked in subconfluent cells treated with cytochalasin D, suggesting that the shift results from merlin dissociation from the actin cytoskeleton, but not from lipid rafts. Intramolecular NH(2)- and COOH-terminal binding, which occurs when merlin transitions to the growth-suppressive form, also brings about a similar change in buoyant density. Our results suggest that constitutive residence of merlin in lipid rafts is crucial for its function and that as merlin becomes growth suppressive in vivo, one significant molecular event may be the loss of interaction with the actin cytoskeleton. To our knowledge, merlin is the first tumor suppressor known to reside within lipid rafts, and the significance of this finding is underscored by known loss-of-function NF2 patient mutations that encode merlins with enhanced TX-100 solubility.

Conformational changes in the hepatitis A virus capsid in response to acidic conditions.

Low pH values encountered during uptake of viruses by receptor-mediated endocytosis have been shown to expose hydrophobic residues of many viruses and result in viral conformational changes leading to uncoating of the viral genome. An assay for hydrophobicity utilising the non-ionic detergent Triton X-114 was established, making use of metabolically-labelled hepatitis A virus (HAV). In this assay, hydrophilic proteins interact with the aqueous (buffer) phase, while hydrophobic proteins interact with the Triton (detergent) phase. HAV particles interact with the aqueous phase at neutral pH, whereas, under acidic conditions, HAV was found predominantly in the detergent phase. This indicates that the capsid of HAV undergoes conformational changes rendering the particle more hydrophobic under acidic conditions. A further two conformational changes were found in HAV on exposure to low pH, as detected by changes in buoyant density in CsCl gradients. These were maturation of provirions to virions and the formation of dense particles. These results may have implications for uncoating of the HAV RNA genome, and these conformational changes could represent intermediates in the viral uncoating process.

Changes of buoyant density during the S-phase of the cell cycle. Direct evidence demonstrated in acute myeloid leukemia by flowcytometry

Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows that the cellular buoyant density increases slightly with up to 0.008 g/ml during the S-phase, at least in cryo-preserved cells used in this study. This contrasts with the generally accepted belief that S-phase cells have a lower or constant buoyant density. A practical implication is that separation of cell (sub)populations based on differences in buoyant density could be flawed to the extent that these populations contain S-phase cells.

Relationship between changes in buoyant density and formation of new sites of cell wall growth in cultures of streptococci (Enterococcus hirae ATCC 9790) undergoing a nutritional shift-up

When the glutamate concentration of cultures of Enterococcus hirae was raised from 20 to 300 micrograms/ml, the mass doubling time decreased from ca. 85 to 45 min in 9 min, but balanced growth was not reestablished for 30 to 40 min. During the unbalanced period of growth, RNA and protein synthesis proceeded more rapidly than did peptidoglycan synthesis, buoyant density increased from ca. 1.1024 to 1.1075 g/ml, and the rate of formation of new cell wall growth sites transitorily accelerated above the new growth rate. When studied as a function of cell size, all cultures showed buoyant density to decrease around cell separation, increase as cells increased in size, and then plateau when cells reached large volumes. Greater increases in buoyant density as a function of cell size were seen after shift-up, with the greatest increases observed at 15 to 20 min after shift-up, when the rate of formation of new sites was also maximal. In a population of cells examined by electron microscopy 15 min after shift-up, buoyant density and the frequency of cells with new sites increased as old sites approached the size of two poles. These data were consistent with a model whereby buoyant density increases in the terminal stages of the cell cycle when the surface grows slower than the cytoplasm. The greater the difference in the rates of inside to outside growth, the greater the increase in buoyant density and the more frequently new sites will be initiated.

A computer model of buoyancy and vertical migration in cyanobacteria

We describe a computer model that simulates diurnal changes in buoyancy and vertical migration by planktonic cyanobacteria in lakes. The model is based on new measurements of the rates of buoyant density change by cyanobacteria in laboratory cultures. The overall rate of density change was found to be the result of two simultaneous processes: (i) a light-dependent increase in density, supported by photosynthesis; and (ii) a time-dependent decrease related to the previous light history. Using the experimentally determined rate constants of these processes, the computer model calculated how buoyant density changes of a colony, responding to the irradiance at a given depth and time of day, would affect its sinking or floating velocity. The model makes a number of interesting predictions on vertical migration, (i) Colony size affects the speed but not the depth range of vertical migrations, (ii) An increase in the proportion of cell volume in a colony has a similar effect to that of colony size, (iii) Surface blooms can be dynamic, with colonies floating up replacing colonies sinking out of the surface bloom, (iv) Phosphorus-limited colonies are likely to migrate to greater depths than light-limited colonies, enabling them to exploit the nutrient-rich depths. The vertical migration of a well-documented natural population is simulated. The model gives a good description of the buoyancy behaviour of the cyanobacterium in the lake. Other simulations explain why colony-forming cells cannot form a stratified layer, while single filaments can. The ecological implications of colony formation are discussed.

Uptake and modification of 125I-lipopolysaccharide by isolated rat Kupffer cells.

While it is generally believed that hepatic clearance of lipopolysaccharide involves Kupffer cells, the mechanism involved has not been fully elucidated. This study assesses this phenomenon in terms of in vitro uptake and post-uptake modification experiments with an 125I-labeled Salmonella minnesota lipopolysaccharide. 125I-Lipopolysaccharide was added to Kupffer cells in suspension cultures under a variety of conditions. In vitro uptake of 125I-Lipopolysaccharide was not saturable up to concentrations of 33.33 micrograms per ml. Kinetics experiments performed at 16.67 micrograms per ml demonstrated that Kupffer cells were unsaturable after 60 min of incubation. The kinetics of uptake could be inhibited, however, by incubation in the presence of a 10-fold excess of unlabeled lipopolysaccharide, indicating that a component of the uptake process may be limited. Energy dependence in this process was demonstrated by incubation in the presence of 1 mM 2-deoxyglucose which inhibited 125I-lipopolysaccharide uptake by approximately 30%. Pretreatment with 7.5 x 10(-5) M colchicine had no effect on kinetics, implying no role for the cell cytoskeleton in lipopolysaccharide uptake. These results are inconsistent with a receptor-mediated process as previously suggested. Modification of internalized label has been demonstrated by changes in buoyant density in CsCl isopyknic density gradients following overnight incubation with Kupffer cells. These results indicate that Kupffer cells clear bacterial endotoxin in vitro and post-uptake degradation occurs within 20 hr of incubation.

Changes in buoyant density and cell size of Escherichia coli in response to osmotic shocks.

The buoyant density of Escherichia coli was shown to be related to the osmolarity of the growth medium. This was true whether the osmolarity was adjusted with either NaCl or sucrose. When cells were grown at one osmolarity and shocked to another osmolarity, their buoyant density adjusted to nearly suit the new osmolarity. When cells were subjected to hyperosmotic shock, they became denser than expected. When cells were subjected to hypoosmotic shock they occasionally undershot the new projected density, but the undershoot was not as dramatic as the overshoot seen with hyperosmotic shocks. Shrinkage and swelling of the cells in response to osmotic shocks could account for the change in their buoyant density. The changes in cell size after osmotic shocks were measured by two independent methods. The first method measured cell size with a Coulter Counter, and the second method measured cell size by stereologic analysis of Nomarski light micrographs. Both methods gave qualitatively similar results and showed the cells to be flexible. The maximum swelling recorded was 23% of the original cell volume, while the maximum shrinkage observed was 33%.

The relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes.

The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with 125I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.

Separation of human natural killer cell subpopulations differentially responsive to interferon potentiation.

Subsets of human natural killer (NK) cells were identified that differed in the capacity to be activated by interferon (IFN) or the IFN-inducer, polyriboinosinic:polyribocytidylic acid [poly(I):poly(C)]. These subsets, which represented effectors of both spontaneous and antibody-dependent cellular cytotoxicity, were physically separable on the basis of cell buoyant density changes induced by exposure of lymphocytes to hyperosmolar Ficoll-Hypaque solutions or by centrifugation of lymphocytes through hyperosmolar (350 mOs/kg) Percoll gradients. Hyperosmolar conditions per se altered neither cell viability, NK cell cytolytic activity, nor the capacity of NK cells in unseparated lymphocyte preparations to be activated by IFN. IFN-unresponsive NK cells, separated by centrifugation through a 350 mOs/kg Percoll layer of 1.069-1.070 g/cm3 specific density, constituted 20 +/- 4% of all active NK cells identified at the single cell level and, per active NK cell, killed comparably to unstimulated IFN-responsive NK cells in 51Cr release assays. Thus, the IFN-unresponsive phenotype was probably not attributable to NK cells that were in an activated state prior to IFN treatment. Surface marker analysis of active NK cells at the single cell level identified comparable proportions in each subfraction to be of the OKM1+, OKT8+, or OKT11+ phenotypes and few, if any, in either subfraction to be of the OKT3+ phenotype. The human IFN-unresponsive NK cell phenotype, in contrast to the corresponding phenotype in the mouse, was therefore not linked to expression of T-cell-associated membrane differentiation antigens.

Instability of brain synaptosomal membrane preparations to repeated ultracentrifugation in isoosmotic density gradients

Synaptosomal membranes from rat brain were found to be remarkably unstable, from the criterion of buoyant density, to repeated density gradient ultracentrifugation. These membranes underwent progressive changes in buoyant density through at least three cycles of fractionation on density gradients generating large proportions of material of both higher and lower bouyant density than the original fraction. The observed density changes were not due to osmotic effects of the various gradient density zones, clumping due to the influence of divalent cations, age of the membrane preparation, length of centrifugation time, intermediate processing steps or a variety of other factors. In addition, the method of homogenization including duration and rate of homogenization had only minimal effects on this properly of buoyant density instability.

Buoyant density changes in the cyanobacterium Microcystis aeruginosa due to changes in the cellular carbohydrate content

The cyanobacterium Microcystis aeruginosa was grown in light-limited chemostat cultures with various light—dark rhythms providing a total periodicity length of 24 h. The buoyant density of the cells changed in parallel with the carbohydrate content. Short incubation experiments with different light intensities, and experiments with the inhibitors iodoacetic acid and arsenate, showed that the buoyant density changes were due to variations in the cellular carbohydrate content. It seems likely that the low dark-growth yield on carbohydrate, which had been stored during the light period, served to facilitate buoyancy changes.

Buoyant density changes in the cyanobacteriumMicrocystis aeruginosadue to changes in the cellular carbohydrate content

The cyanobacterium Microcystis aeruginosa was grown in light-limited chemostat cultures with various light—dark rhythms providing a total periodicity length of 24 h. The buoyant density of the cells changed in parallel with the carbohydrate content. Short incubation experiments with different light intensities, and experiments with the inhibitors iodoacetic acid and arsenate, showed that the buoyant density changes were due to variations in the cellular carbohydrate content. It seems likely that the low dark-growth yield on carbohydrate, which had been stored during the light period, served to facilitate buoyancy changes.

Response of rhesus serum high density lipoproteins to cycles of diet-induced hypercholesterolemia.

Two male rhesus monkeys underwent cyclical feeding of a hypercholesterolemic diet (2% cholesterol, 25% coconut oil) and a low-fat Purina monkey chow diet. During the latter diet, high density lipoprotein (HDL) exhibited two components with peak densities of d = 1.081 g/ml and 1.109 g/ml named HDLL and HDLH, respectively. During the initial hypercholesterolemic stage, except for apo A-II which remained unchanged, there was a transient rise in HDL (mainly HDLL) as well as in HDL cholesterol and apo A-I, all reaching maximal values after about 2 weeks from the onset of the diet. The two HDL species changed neither in size nor density as compared to their baseline counterparts, but had a comparatively higher content in cholesteryl ester and lesser amounts of triglycerides and phospholipids as compared to the normocholesterolemic animal. With the development of overt hypercholesterolemia (plasma cholesterol levels above 400 mg/dl), both HDL particles increased in density due to the loss of surface components (phospholipids and unesterified cholesterol) and core triglycerides with only minor changes in protein and cholesteryl ester contents. At this stage, the same two animals exhibited significant changes in the size and buoyant density of LDL. When returned to a normal Purina chow diet, the animals' serum cholesterol levels declined rapidly to normal levels; normalization of the HDL distribution also occurred but at a comparatively later time (26 weeks). Our studies indicate that the two HDL subsets characteristic of the normocholesterolemic rhesus monkey undergo significant changes in buoyant density as a function of the stage of hypercholesterolemia and that changes in concentration and size mainly affect the HDLL subspecies. At levels of plasma cholesterol below 400 mg/dl, this cholesterol increment is reflected by a significant increase in the number of the HDL subspecies without the overt participation of the low density lipoprotein classes characteristic of the advanced hyperlipidemic stage. Since we previously reported that greatly increased levels of cholesteryl esters enriched low density lipoproteins, beta-VLDL (very low density lipoprotein) and pre-beta-VLDL during overt diet-induced hypercholesterolemia, it is apparent that cholesterol is distributed differently among lipoprotein particles containing either apo A-I, apo B, or apo E depending on its concentration in plasma.

Interaction of a DNA-binding protein, the gene product of D5 of bacteriophage T5, with double-stranded DNA. Analysis by metrizamide gradient centrifugation.

Interactions of DNA and the gene product D5 (gpD5) of bacteriophage T5, a DNA-binding protein that binds preferentially and cooperatively to double-stranded DNA, were analyzed by metrizamide gradient centrifugation. Conditions were set so that DNA and DNA protein complex sedimented to apparent equilibrium positions. DNA has a buoyant density of 1.12 g/cm3, and DNA saturated with gpD5 has a buoyant density of 1.17 g/cm3. These values are independent of DNA size and base composition in the range studied. At gpD5 concentration below the saturation value in a low ionic strength buffer, DNA distribution is bimodal, indicating cooperative binding of gpD5 to DNA. However, in the presence of 10 mM MgCl2, the binding process becomes distributive, with the buoyant density increasing linearly with the amount of gpD5 added until the saturation. From these data, one molecule of gpD5 is calculated to cover 40 base pairs at saturation. The technique as described has general applicability to the study of any interaction between DNA and dNA-binding proteins that bind in sufficient amount to cause detectable changes in buoyant density.

Preparation of Avocado Mitochondria Using Self-Generated Percoll Density Gradients and Changes in Buoyant Density during Ripening

Mitochondria from avocado (Persea americana Mill, var. Fuerte and Hass) can be rapidly prepared at every stage of ripening using differential centrifugation and self-generated Percoll gradients. The procedure results in improved oxidative and phosphorylative properties, especially for mitochondria isolated from preclimacteric fruits.A gradual change in the buoyant density of avocado mitochondria takes place during ripening. Climacteric and postclimacteric avocado mitochondria have the same buoyant density as other plant mitochondria (potato, cauliflower), whereas mitochondria from preclimacteric fruit have a lower density. The transition in buoyant density occurs during the climacteric rise, and two populations of intact mitochondria (p = 1.060 and p = 1.075) can be separated at this stage. Evidence indicates that the difference in mitochondrial buoyant density between preclimacteric and postclimacteric mitochondria is likely due to interactions with soluble cytosolic components.

Analysis of buoyant density of canine peripheral blood leukocytes with PVP-Silica (Percoll) density gradients

Isolation of canine peripheral blood mononuclear cells with a one step centrifugal separation procedure has not been very succesful sofar. Significant contamination with polymorphonuclear cells has been reported. An analysis of the buoyant density of canine peripheral blood leukocytes on a self-generating Percoll gradient showed that the buoyant densities of polymorphonuclear cells and lymphocytes are so near that separation with high purity and yield is not possible with the use of a density gradient. Transient changes in buoyant density of polymorphonuclear cells have been observed. In such situations differences in buoyant density between cell types have been observed which permit separation of mononuclear cells from polymorphonuclear cells at a reasonable yield.

Modification of PM2 DNA with N-acetoxy-2-acetylaminofluorene: Changes in buoyant density, electrophoretic mobility and electron microscopic structure

PM2 DNA, forms I and II, were chemically modified with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene. The products were subjected to 3 analytical procedures: (i) buoyant density centrifugation in the presence of the intercalating dyes ethidium bromide or propidium diiodide; (ii) agarose gel electrophoresis: and (iii) electron microscopy. It was found that carcinogen-modified DNAs banded at higher densities in the presence of either dye. This effect was far more pronounced with form I DNA than with form II. Form I DNA showed a concentration-dependent decrease of migration velocity in agarose gel electrophoresis after modification; form II DNA exhibited a similar but smaller effect. Electron microscopy of form I DNA did not reveal a consistent correlation between the extent of chemical modification and the loss of superhelical turns. Form II DNA exhibited characteristic alterations such as aggregation of several PM2 molecules into network-like structures, kinky configuration, and probably single-stranded bubbles extending over 600–1500 bases.

The modification of biophysical and endotoxic properties of bacterial lipopolysaccharides by serum.

Normal rabbit serum reduces the buoyant density of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (d = 1.44 g/cm3) and Salmonella minnesota R595 (d = 1.38 g/cm3) to a value less than g/cm3. This density shift is associated with the inhibition of a number of endotoxic activities of the LPS; namely, the pyrogenic activity, the ability to produce an immediate neutropenia in rabbits, lethality in adrenalectomized mice, and anticomplementary activity. A qualitatively similar change in buoyant density was observed to occur after intravenous injection of the LPS into rabbits. Preliminary evidence suggests that the density shift does not occur as a result of the degradation of the glycolipid backbone of the LPS. These data suggest that the interactions of LPS with plasma (or serum) components leading to reduction in buoyant density may account for a major pathway of LPS detoxification.