Role of monocyte colony-stimulating factor in foam cell generation.

Recently, we reported that human monocyte colony-stimulating factor (M-CSF) stimulates the clearance of lipoproteins containing apoB100 via both low density lipoprotein receptor-dependent and -independent pathways in target cells of M-CSF, and reduces plasma cholesterol level (Journal of Biological Chemistry, 265:12869-12875, 1990). This suggests a linkage of cytokines to the metabolic regulation of plasma cholesterol. Furthermore, we found a significant role of M-CSF in cholesterol metabolism of human monocyte-derived macrophages. M-CSF enhanced not only the uptake of acetylated low density lipoprotein and oxidized low density lipoprotein in macrophages, but also the efflux of cholesterol from cholesterol-loaded macrophages. To elucidate in vivo effects of M-CSF on cholesterol efflux from tissues, we administered an intravenous injection of 3H-cholesterol (150 microCi) into WHHL rabbits 1 month before starting M-CSF treatment. We observed an increased cholesterol efflux from tissues to plasma high density lipoprotein after M-CSF treatment when cholesterol efflux was estimated as the change in specific radioactivity of plasma high density lipoprotein-cholesterol. This result suggests that M-CSF can enhance the excretion of cholesterol from target cells of M-CSF, such as cholesterol-loaded macrophages in the arterial wall, and reduce the rate of atherogenesis.     

Intracellular generation of reactive oxygen species by contracting skeletal muscle cells

The aim of this work was to examine the intracellular generation of reactive oxygen species in skeletal muscle cells at rest and during and following a period of contractile activity. Intracellular generation of reactive oxygen species was examined directly in skeletal muscle myotubes using 2',7'-dichlorodihydrofluorescein (DCFH) as an intracellular probe. Preliminary experiments confirmed that DCFH located to the myotubes but was readily photoxidizable during repeated intracellular fluorescence measurements and strategies to minimize this were developed. The rate of oxidation of DCFH did not change significantly over 30 min in resting myotubes, but was increased by approximately 4-fold during 10 min of repetitive, electrically stimulated contractile activity. This increased rate was maintained over 10 min following the end of the contraction protocol. DCF fluorescence was distributed evenly throughout the myotube with no evidence of accumulation at any specific intracellular sites or localization to mitochondria. The rise in DCF fluorescence was effectively abolished by treatment of the myotubes with the intracellular superoxide scavenger, Tiron. Thus these data appear to represent the first direct demonstration of a rise in intracellular oxidant activity during contractile activity in skeletal muscle myotubes and indicate that superoxide, generated from intracellular sites, is the ultimate source of oxidant(s) responsible for the DCFH oxidation.     

Intracellular localization of hyaluronan in proliferating cells.

Hyaluronan is a high molecular weight glycosaminoglycan found in the extracellular matrix of many tissues, where it is believed to promote cell migration and proliferation. It was recently shown that hyaluronan-dependent pericellular matrix formation is a rapid process that occurs as cells detach during mitosis. Growing evidence for intracellular hyaluronan in tissues in vivo, together with evidence of intracellular hyaluronan binding molecules, prompted us to examine hyaluronan distribution and uptake as well as hyaluronan binding sites in cells and their relationship to cell proliferation in vitro, using a biotinylated hyaluronan binding protein and fluorescein-labeled hyaluronan. In permeabilized smooth muscle cells and fibroblasts, hyaluronan staining was seen in the cytoplasm in a diffuse, network-like pattern and in vesicles. Nuclear hyaluronan staining was observed and confirmed by confocal microscopy and was often associated with nucleoli and nuclear clefts. After serum stimulation of 3T3 cells, there was a dramatic increase in cytoplasmic hyaluronan staining, especially during late prophase/early prometaphase of mitosis. In contrast, unstimulated cells were negative. There was a pronounced alteration in the amount and distribution of hyaluronan binding sites, from a mostly nucleolar distribution in unstimulated cells to one throughout the cytoplasm and nucleus after stimulation. Exogenous fluorescein-labeled hyaluronan was taken up avidly into vesicles in growing cells but was localized distinctly compared to endogenous hyaluronan, suggesting that hyaluronan in cells may be derived from an intracellular source. These data indicate that intracellular hyaluronan may be involved in nucleolar function, chromosomal rearrangement, or other events in proliferating cells. (J Histochem Cytochem 47:1331-1341, 1999)     

Improved power generation using nitrogen-doped 3D graphite foam anodes in microbial fuel cells

Bioprocess and Biosystems Engineering - The properties of the anode material and structure are critical to the microbial growth and interfacial electron transfer between the biofilm and the anode....     

Intracellular topology and epitope shielding of poliovirus 3A protein

The poliovirus RNA replication complex comprises multiple viral and possibly cellular proteins assembled on the cytoplasmic surface of rearranged intracellular membranes. Viral proteins 3A and 3AB perform several functions during the poliovirus replicative cycle, including significant roles in rearranging membranes, anchoring the viral polymerase to these membranes, inhibiting host protein secretion, and possibly providing the 3B protein primer for RNA synthesis. During poliovirus infection, the immunofluorescence signal of an amino-terminal epitope of 3A-containing proteins is markedly shielded compared to 3A protein expressed in the absence of other poliovirus proteins. This is not due to luminal orientation of all or a subset of the 3A-containing polypeptides, as shown by immunofluorescence following differential permeabilization and proteolysis experiments. Shielding of the 3A epitope is more pronounced in cells infected with wild-type poliovirus than in cells with temperature-sensitive mutant virus that contains a mutation in the 3D polymerase coding region adjacent to the 3AB binding site. Therefore, it is likely that direct binding of the poliovirus RNA-dependent RNA polymerase occludes the amino terminus of 3A-containing polypeptides in the RNA replication complex.     

Intracellular freezing of glycerolized red cells.

The response of glycerolized human red blood cells to freezing has been evaluated in terms of the thermodynamic state of the frozen intracellular medium. The physiochemical conditions requisite for intracellular freezing, characterized by the cooling rate and the degree of extracellular supercooling, are altered appreciably by the prefreezing addition of glycerol to the cells.Fresh human erythrocytes were suspended in an isotonic glycerol solution yielding a final cryophylactic concentration of either 1.5 or 3.0 m. Subsequently the cell suspension was frozen on a special low temperature stage, mounted on a light microscope, at controlled constant cooling rates with varying degrees of extracellular supercooling (ΔT_sc). The formation of a pure intracellular ice phase was detected by direct observation of the cells.The addition of glycerol produced several significant variations in the freezing characteristics of the blood. As in unmodified cells, the incidence of intracellular freezing increased with the magnitudes of both the cooling rate and the extracellular supercooling. However, the glycerolized cells exhibited a much greater tendency to supercool prior to the initial nucleation of ice. Values of ΔT_sc > ?20 °C were readily obtained. Also, the transition from 0 to 100% occurrence of intracellular ice covered a cooling rate spectrum in excess of 300 to 600 °K/min, as compared with 10 °C/min for unmodified cells. Thus, the incidence of intracellular ice formation was significantly increased in glycerolized cells.     

Intracellular chloride activity in cultured mesangial cells.

Glomerular mesangial cells require Cl ions for the development of a variety of metabolic and functional properties. In the present study the electrochemical distribution for Cl- was examined in cultured rat mesangial cells with Cl(-)-sensitive intracellular microelectrodes. It was determined that the intracellular Cl activity exceeded the levels predicted for a passively distributed ion. This was further substantiated by exposing mesangial cells to 10(-5) M bumetanide which drove intracellular Cl to a value close to electrochemical equilibrium. We conclude that Cl accumulates in mesangial cells, against its electrochemical gradient, through a transport pathway that is highly sensitive to bumetanide.     

Intracellular site of gamma-secretase cleavage for Abeta42 generation in neuro 2a cells harbouring a presenilin 1 mutation.

Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid beta-protein (Abeta)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Abeta42-gamma-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Abeta42-gamma-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP-C100 encoding the C-terminal beta-amyloid precursor protein (APP) fragment truncated at the N terminus of Abeta (C100); C100 requires only gamma-secretase cleavage to yield Abeta. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced Abeta1-42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of Abeta1-40 was not increased. The intracellular generation of Abeta1-42 from transiently expressed C100 in both mutated PS1-induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Abeta1-42 and Abeta1-40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of gamma-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Abeta1-42/40 from C100 was not influenced by monensin treatment, and the level of Abeta1-42/40 generated from C100 carrying a sorting signal for the trans-Golgi network was higher than that generated from wild-type C100. These results using PS1-mutation-harbouring and wild-type Neuro 2a cells suggest that Abeta42/40-gamma-secretase cleavages occur in the Golgi compartment and the trans-Golgi network, and that the PS1 mutation does not alter the intracelluar site of Abeta42-gamma-secretase cleavage in the normal APP proteolytic processing pathway.     

Intracellular mechanisms of gonadotropin-stimulated gene expression in granulosa cells.

Previous studies have shown that the gonadotropins follicle-stimulating hormone and luteinizing hormone stimulate proopiomelanocortin (POMC) promoter activity and mRNA levels in ovarian granulosa cells. The objective of these studies was to determine the role of cAMP-dependent protein kinases (pKA) in gonadotropin-stimulated gene expression. Primary cultures of rat granulosa cells were transfected with a gene construct consisting of the POMC promoter (-150 to +63; designated pOMC-CAT) fused to the chloramphenicol acetyltransferase (CAT) reporter gene either alone or cotransfected with an expression plasmid (designated mutant RI), which overexpresses a mutant form of the murine RI subunit incapable of binding cAMP and serving as an irreversible inhibitor of the catalytic subunit of pKA. Follicle-stimulating hormone or isoproterenol caused a significant stimulation of pOMC-CAT activity in transfected cells. Cotransfection of pOMC-CAT with mutant RI caused a significant inhibition of basal pOMC-CAT activity and abolished the gonadotropin stimulation. As a control, transfection of the SV-40 viral enhancer-promoter fused to CAT (pSV2-CAT) was unresponsive to follicle-stimulating hormone stimulation and cotransfection with mutant RI had no significant effect on pSV2-CAT activity. These studies suggest that gonadotropin regulation of the POMC promoter is mediated by pKA and that promoter activity is stringently controlled by pKA.     

Intracellular distribution of lipophilic fluorescent probes in mammalian cells.

The intracellular distribution of several hydrophobic fluorescent probes (1,6-diphenyl-1,3,5-hexatriene (DPH), perylene, and 2-p-toluidinyl-6-naphthalene sulfonate (TNS) in mouse lymphocytes and a fibroblast cell line was examined using radiolabeled fluorescent probes and the technique of high resolution EM autoradiography. Following a short term incubation, DPH and perylene were found largely internalized in cells, while TNS was localized predominantly at the cell surface. These findings suggest that fluorescence polarization studies using such probes with intact cells do not necessarily monitor only the cell surface membrane and must be interpreted with caution.     

Intracellular degradation of glycoproteins in Acer pseudoplatanus L. cells

The degradation of endogenously labelled glycoproteins was studied in Acer pseudoplatanus L. cell suspension cultures in experiments using a dual-label with [¹⁴C]mannose and [³H]leucine.After harvesting the cells, protoplasts were prepared and vacuoles isolated. More than 30% of both total newly synthesized proteins (³H radioactivity) and glycoproteins (¹⁴C radioactivity) were recovered inside the vacuoles, the lytic compartment of plant cells. Half of these proteins were degraded when isolated vacuoles were incubated for 6 h at 20°C. So, the vacuolar compartment appears to be a major site of glycoprotein degradation in the cell.The glycoproteins were degraded at the same rate as the total newly synthesized proteins. However, some vacuolar hydrolytic enzymes were found to be glycoproteins and resistant to proteolytic attack. The biochemical explanation for such a resistance is not clear at this time, but in Acer cells the presence of covalently bound carbohydrates in proteins does not seem to be involved in the selectivity of protein turnover.     

Intracellular targeting of calmodulin mRNAs in primary hippocampal cells.

We investigated the intracellular distribution of the mRNAs corresponding to the three non-allelic CaM genes in cultured hippocampal cells by in situ hybridization with digoxigenin-labeled gene-specific riboprobes. In neurons the perikaryon was heavily stained and strong dendritic mRNA targeting was detected for all three CaM genes. The color labeling exhibited a punctate distribution, suggesting that CaM mRNAs are transported in RNA granules. Immunocytochemistry for S100 demonstrated that glial cells express CaM mRNAs at a very low level. A minority of the cultured cells were negative for either labeling.     

Intracellular activity in cricket neurons during generation of song patterns

Summary During production of song patterns by the semi-isolated CNS of Gryllus campestris, intracellullar recordings were made in fibers of the mesothoracic ganglion, including synaptic areas of identified wing opener and closer motor neurons. The normal calling song pattern and some transitional songs toward courtship and toward aggression were generated by the CNS in the absence of any phasic sensory timing (Figs. 1, 4). Intracellular activity of the opener motor neurons was characterized by an absence of events in the interchirp interval, an EPSP underlying each burst, and an IPSP following the burst if the closer motor neurons were to be activated (Fig. 1). Intracellular activity of the closer motor neurons was characterized by an absence of events in the interchirp interval, an IPSP immediately following the onset of the opener motor neuron burst, and an EPSP after the IPSP (Figs. 2, 3). Units were found which fired in a burst during the period when both the opener and closer motor neurons were inhibited (Fig. 5). Complementary sets of units were found which displayed an oscillation of activity at the chirp rhythm but not at the pulse rhythm (Fig. 6). Gaps in the calling song were observed whose characteristics indicated that motor neuron activity was neither required for, nor effective in, resetting the chirp timing oscillator (Fig. 8). A possible model for the song generating mechanism is outlined.     

Cultivation of animal cells in a reticulated vitreous carbon foam.

A reticulated vitreous carbon foam (RVCF) was used as a surface to cultivate a model anchorage-dependent animal cell line, 3T6 (mouse embryo fibroblast). This fixed-surface bioreactor provided a low-shear, chemically-inert, and reusable environment for cell growth. An external medium recirculation loop allowed aeration, nutrient monitoring, and medium replacement without disturbing the cells. Optimal flow rates for the attachment and growth phases were determined. Growth rates comparable to static (T-flask and petri dish) cultures and agitated microcarrier cultures were achieved with appropriately high medium recirculation rates. Metabolic parameters were shown to be useful indicators of cell mass, although specific glucose consumption rates were considerably higher for cultures in the RVCF reactor. Oxygen supply was shown to be the most likely limiting factor for scaleup.     

Intracellular distribution of endothelin-1 receptors in rat liver cells.

We studied the binding of (125I)-endothelin-1 as well as that of the vasopressin analogue (125I)-[8-phenylpropionyl]-LVP to purified plasma membranes, Golgi cisternae and cell nuclei from rat liver. Cell organelles were isolated by differential centrifugation and discontinuous sucrose gradients. Endothelin-1 exhibited specific binding to plasma membranes, Golgi cisternae and nuclei, while the binding of (125I)-[8-phenylpropionyl]-LVP was restricted to the plasma membranes. The number of receptors (Bmax) and the binding constants (Kd) were determined by Scatchard analysis of competition binding studies. In all cases only one class of Et-1 binding sites could be detected. The presence of Et-1 receptors on the Golgi complex either indicates that the receptor is glycosylated within the cisternae or alternatively, there exists a recycling pathway. The unexpected finding of Et-1 receptors on highly purified nuclei suggests that this peptide may exert part of its biological functions intracellularly via the nucleus.     

Intracellular transport of endocytosed proteins in rat liver endothelial cells.

1. Receptor-mediated endocytosis of mannose-terminated glycoproteins in rat liver endothelial cells has been followed by means of subcellular fractionation and by immunocytochemical labelling of ultrathin cryosections after intravenous injection of ovalbumin. For subcellular-fractionation studies the ligand was labelled with 125-tyramine-cellobiose adduct, which leads to labelled degradation products being trapped intracellularly in the organelle where the degradation takes place. 2. Isopycnic centrifugation in sucrose gradients of a whole liver homogenate showed that the ligand is sequentially associated with three organelles with increasing buoyant densities. The ligand was, 1 min after injection, recovered in a light, slowly sedimenting vesicle and subsequently (6 min) in larger endosomes. After 24 min the ligand was recovered in dense organelles, where also acid-soluble degradation products accumulated. 3. Immunocytochemical labelling of ultrathin cryosections showed that the ligand appeared rapidly after internalization in coated vesicles and subsequently in two larger types of endosomes. In the 'early' endosomes (1 min after injection) the labelling was seen closely associated with the membrane of the vesicle; after 6 min the ligand was evenly distributed in the lumen. At 24 min after injection the ligand was found in the lysosomes. 4. A bimodal distribution of endothelial cell lysosomes with different buoyant densities was revealed by centrifugation in iso-osmotic Nycodenz gradients, suggesting that two types of lysosomes are involved in the degradation of mannose-terminated glycoproteins in liver endothelial cells. Two populations of lysosomes were also revealed by sucrose-density-gradient centrifugation after injection of large amounts of yeast invertase. 5. In conclusion, ovalbumin is transferred rapidly through three endosomal compartments before delivering to the lysosomes. The degradation seems to take place in two populations of lysosomes.     

Phosphatidylserine-mediated delivery of cholesterol to macrophages: a novel experimental method for the generation of foam cells

P388D1 macrophages were incubated for 24 h with cholesterol-phosphatidylserine liposomes (50 micrograms cholesterol/ml) and the content of cellular cholesteryl esters increased to approx. 200 micrograms/mg cell protein. Similar results were not observed with cholesterol-phosphatidylcholine liposomes. These results demonstrate that specific phospholipid-cholesterol liposomes can be utilized for the experimental production of macrophage cholesterol-rich foam cells.     

Intracellular pH in singleSaccharomyces cerevisiae cells

Saccharomyces cerevisiae cells can be stained with the pH dependent fluorochrome BCECF, to monitor shifts in intracellular pH in individual cells using flow cytometry. Cells stained in the presence of ethanol were found to be stained as much as 100 times more intensely as cells without ethanol. Cells that have been starved for the carbon source showed a significant shift in intracellular pH upon refeeding with a metabolizable carbon source within 3 minutes.