Mg Of Cell Protein Research Articles

Studies on the anaerobic degradation of benzoic acid and 2-aminobenzoic acid by a denitrifying Pseudomonas strain

The growth of a denitrifying Pseudomonas strain on benzoic acid and 2-aminobenzoic acid (anthranilic acid) has been studied. The organism grew aerobically on benzoate, 2-aminobenzoate, and gentisate, but not on catechol or protocatechuic acid. These and other findings suggest that aerobic degradation of benzoic acid was via gentisic acid. Under completely anaerobic conditions in the presence of nitrate, benzoate and 2-aminobenzoate (5 mM each) were oxidized to CO2 with the concurrent reduction of NO3-to NO2-. Only after complete NO3-consumption was NO2-reduced to N2. Cells contained a NADP-specific 2-oxoglutaate dehydrogenase, in contrast to a NAD-specific pyruvate dehydrogenase. During anaerobic metabolism of [carboxyl-14C]benzoic acid, 16% of the label of metabolized benzoic acid was incorporated into cell material; this excludes intermediary decarboxylation during anaerobic metabolism. Extracts catalysed the activation of benzoic acid and a variety of its derivatives to the respective aryl-coenzyme A thioesters, ATP being cleaved to AMP and PPi; two synthetase activites were present. Extracts from 2-aminobenzoate-grown cells catalyzed a NADH-dependent reduction of 2-aminobenzoyl-CoA (100 nmol·min-1·mg-1 cell protein) to an unidentified CoA thioester, with a stoichiometric release of NH3 and a stoichiometry of ≈ 3 mol NADH oxidized per mol 2-aminobenzyol-CoA reduced when tested under aerobic conditions. The 2-aminobenzoyl-CoA reductase activity was lacking in anaerobic benzoate-grown cells and in aerobic cells. This is taken as evidence that 2-aminobenzoyl-CoA reductase is a key enzyme in a novel reductive pathway of anaerobic 2-aminobenzoic acid metabolism.

Glycolytic flux in Zymomonas mobilis: enzyme and metabolite levels during batch fermentation.

The rate at which Z. mobilis (Entner-Doudoroff pathway) converts high concentrations of glucose (20%) into ethanol plus CO2 changes as ethanol accumulates in the surrounding broth. This decline in glycolytic activity (per milligram of cell protein) does not result from inhibitory effects of ethanol, which can be reversed immediately by ethanol removal. The peak of fermentative activity (58 mumol of CO2 evolved per mg of cell protein per h) occurred after the accumulation of 1.1% ethanol (18 h) and declined to one-half this rate after 30 h (6.2% accumulated ethanol), although the cell number continued to increase. These times corresponded to the end of exponential growth and to the onset of the stationary phase (on the basis of measurement of cell protein), respectively. An examination of many of the requirements for fermentation (nucleotides, magnesium, enzyme levels, intracellular pH, delta pH) revealed three possible reasons for this early decline in activity: decreased abundance of nucleotides, a decrease in internal pH from 6.3 to 5.3, and a decrease in the specific activities of two glycolytic enzymes (pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase). 31P nuclear magnetic resonance spectra of perchlorate extracts from cells fermenting in broth revealed very low levels of glycolytic intermediates (Entner-Doudoroff pathway) in cells examined at the peak of fermentative activity (18-h cells) in comparison with cells examined at a later stage (30-h cells), consistent with limitation of the fermentation rate by glycolytic enzymes near the end of the pathway. It is likely that cell death (loss of colony-forming ability) and the collapse of delta pH also contribute to the further decline in fermentative activity after 30 h.

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.

The interaction in vivo of transferrin and asialotransferrin with liver cells

Rat transferrin or asialotransferrin doubly radiolabelled with 59Fe and 125I was injected into rats. A determination of extrahepatic and hepatic uptake indicated that asialotransferrin delivers a higher fraction of the injected 59Fe to the liver than does transferrin. In order to determine in vivo the intrahepatic recognition sites for transferrin and asialotransferrin, the liver was subfractionated into parenchymal, endothelial and Kupffer cells by a low-temperature cell isolation procedure. High-affinity recognition of transferrin (competed for by an excess of unlabelled transferrin) is exerted by parenchymal cells as well as endothelial and Kupffer cells with a 10-fold higher association (expressed per mg of cell protein) to the latter cell types. In all three cell types iron delivery occurs, as concluded from the increase in cellular 59Fe/125I ratio at prolonged circulation times of transferrin. It can be calculated that parenchymal cells are responsible for 50-60% of the interaction of transferrin with the liver, 20-30% is associated with endothelial cells and about 20% with Kupffer cells. For asialotransferrin a higher fraction of the injected dose becomes associated with parenchymal cells as well as with endothelial and Kupffer cells. Competition experiments in vivo with various sugars indicated that the increased interaction of asialotransferrin with parenchymal cells is specifically inhibited by N-acetylgalactosamine whereas mannan specifically inhibits the increased interaction of asialotransferrin with endothelial and Kupffer cells. Recognition of asialotransferrin by galactose receptors from parenchymal cells or mannose receptors from endothelial and Kupffer cells is coupled to active 59Fe delivery to the cells. It is concluded that, as well as parenchymal cells, liver endothelial and Kupffer cells are also quantitatively important intrahepatic sites for transferrin and asialotransferrin metabolism, an interaction exerted by multiple recognition sites on the various cell types.

Growth hormone binding and stimulation of amino acid uptake in epithelial cells of rat ventral prostate.

The binding of [125I]-human growth hormone (hGH) was studied in epithelial cells isolated from rat ventral prostate. Binding and degradation were dependent on time and temperature. The effect of a lysosomotropic agent suggested internalization and lysosomal degradation of the hormone. Dissociation and stoichiometric studies indicated the existence of a single class of GH receptors with a Kd of 0.7 nM and a binding capacity of 46 fmol hGH bound mg-1 cell protein. The receptor appeared to possess a somatotrophic nature since lactogenic hormones such as human placental lactogen and rat prolactin exhibited a very low degree of competition (whereas a variety of unrelated hormones and neuropeptides showed no effect). GH-stimulated leucine uptake by the cells in a time- and dose-dependent manner, half maximal effect being observed at 0.32 nM GH thus suggesting a direct relationship with the binding step.

Stereoselective uptake of the GABA-transaminase inhibitors gamma-vinyl GABA and gamma-acetylenic GABA into neurons and astrocytes.

The cellular uptake of the GABA-transaminase inhibitors gamma-vinyl GABA (GVG) and gamma-acetylenic GABA (GAG) was studied in cultured neurons and astrocytes. By the use of the individual enantiomers R- and S-GVG and R- and S-GAG it could be shown that in both cell types only the S-enantiomers could be actively transported. Comparing neurons and astrocytes only neurons exhibited a high affinity uptake system for S-GVG (Km 78.2 +/- 20.3 microM; Vmax 0.71 +/- 0.06 nmol.min-1.mg-1 cell protein). In case of S-GAG it could not be established with certainty whether the neuronal uptake was of the high affinity type. Both GVG and GAG were studied as inhibitors of GABA uptake into neurons and astrocytes. S-GVG and S-GAG were found to be weak inhibitors of GABA uptake suggesting that S-GVG is not transported by the GABA carrier in neurons. The finding of a much more efficient uptake of S-GVG into neurons than into astrocytes is in line with the previous observation that neuronal GABA-T is more sensitive than astrocytic GABA-T to S-GVG.

Inositol 1,4,5-trisphosphate and intracellular Ca2+ homeostasis in clonal pituitary cells (GH3). Translocation of Ca2+ into mitochondria from a functionally discrete portion of the nonmitochondrial store.

Ca2+-specific minielectrodes were used to monitor changes in the ambient free Ca2+ concentration [( Ca2+]a) maintained by the intracellular organelles of permeabilized GH3 cells. Mitochondria maintained a [Ca2+]a steady state of around 500 nM and displayed a very high capacity for Ca2+ uptake. A nonmitochondrial pool, tentatively identified as the endoplasmic reticulum (ER), displayed higher affinity for Ca2+ by maintaining a steady state of approximately 170 nM. The capacity of this pool was around 10 nmol/mg cell protein. Inositol 1,4,5-trisphosphate (InsP3) released Ca2+ specifically from the ER, with an EC50 of approximately 2 microM, and gave maximal release of around 4 nmol Ca2+/mg of cell protein. Repeated InsR3 additions under conditions allowing for functional mitochondrial transport resulted in successively attenuated peaks, leading eventually to the depletion of the InsP3 sensitive portion of the ER. However, Ca2+ could still be released from the total ER pool with the ATPase inhibitor, vanadate. This InsP3-insensitive store did not reaccumulate InsP3 releasable Ca2+ nor could it directly refill the sensitive pool. However, the attenuation of the InsP3 responses could be overcome by repleting the sensitive pool with exogenous Ca2+ or by inhibiting Ca2+ uptake into the mitochondria. The results suggest: 1) the ER is the major intracellular organelle buffering Ca2+ in nonstimulated GH3 cells; 2) InsP3 releases Ca2+ from only a portion of the ER; 3) the InsP3-sensitive and -insensitive ER pools are functionally distinct; 4) InsP3 addition results in a transfer of Ca2+ from the ER to the mitochondria.

Contraction of collagen lattice by peritubular cells from rat testis.

Peritubular cells from 15- and 25-day-old rat testis trapped in collagen lattices caused those lattices to contract. Contraction proceeded more rapidly and to a greater extent using cells from younger rats. When 36,000 cells from 15- and 25-day-old rats were trapped in 800 mm2 lattices, the areas were reduced to 28 mm2 and 170 mm2, respectively, within 24 h. The cells from older rats were less effective at contracting the lattice than cells from younger rats. Cytochalasin B (5 micrograms ml-1) inhibited lattice contraction and caused disruption of actin filaments as seen by fluorescent staining with Rh-phalloidin. Cholera toxin (10 micrograms ml-1), and 1 mM-dibutyryl cAMP inhibited lattice contraction, as did 10 microM-trifluoperazine, commonly an inhibitor of calmodulin. The total intracellular concentration of cAMP was greater in peritubular cells from 25-day-old rats than in those from 15-day-old rats: 427 +/- 34 and 120 +/- 16 pmol mg-1 cell protein, respectively. When peritubular cells in monolayer were permeabilized with glycerol, the addition of ATP caused the cells to contract. Cell contraction was greater in cells from 15-day-old rats than 25-day-old rats. When cells were grown on silicone rubber, they caused that surface to wrinkle. Peritubular cells from 15-day-old rats caused the onset of wrinkling at 4 h. At the same time, no wrinkling was observed with cells from 25-day-old rats. Studies of lattice contraction and cell contraction were also made using cells from 20-day-old rats. In each case, contraction was intermediate between that of cells from 15-and 25-day-old rats. The possibility exists that lattice contraction, cell contraction and wrinkling of silicone film result from a mechanism of actin filament sliding, generated by myosin ATPase activity, and is inhibited by cAMP. The reduced rate of contraction in cells from 25-day-old rats may be related to their higher intracellular levels of cAMP. Evidence exists to show that cAMP blocks myosin ATPase activity by inhibiting the phosphorylation of its regulatory peptide, myosin light chain.

Physicochemical transfer of [3H]cholesterol from plasma lipoproteins to cultured human fibroblasts.

The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.

Changes in protein synthesis and breakdown rates and responsiveness to growth factors with ageing in human lung fibroblasts

The effects of insulin, epidermal growth factor, insulin-like growth factor-2 and fetal bovine serum on protein synthesis and protein breakdown have been measured in mid-passage and senescent cultures of human diploid lung fibroblasts. Each of the individual growth factors was a potent inhibitor of protein breakdown with no difference in either the maximum response or sensitivity evidence between senescent cells and mid-passage cultures. Binding of 125I-labelled epidermal growth factor per mg of cell protein similarly showed no difference between senescent and confluent mid-passage fibroblasts, although sparse mid-passage cells bound more of the ligand. These results indicate normal binding and normal responsiveness to growth factors in senescent cultures. However, rates of protein synthesis are higher in confluent mid-passage cells and especially so in sparse mid-passage cells than in senescent cells of intermediate density. Furthermore, senescent cells differ from either growth state of mid-passage cells because protein synthesis in aged cells is much less responsive to any of the growth factors or to fetal bovine serum. It is suggested from these results that the reduced ability of serum or growth factors to initiate DNA synthesis or growth in ageing cells may be a consequence of an unresponsive protein synthesis pathway rather than a generalised defect in growth factor action.

Aryl hydrocarbons as inducers of cytochrome P-450 in Acinetobacter calcoaceticus

The inducibility of cytochrome P-450 in Acinetobacter calcoaceticus by some compounds known as typical inducers of hepatic cytochromes P-450 was investigated. Besides biphenyl also indene and phenanthrene are inducers, whereas compounds of the so-called “phenobarbital type” are not. Biphenyl appears to be the most effective inducer with regard to the yield of cytochrome P-450/mg of cell protein. By addition of the compounds in the vapour phase an induction of the protein by naphthalene could be demonstrated. The results are indicative of the existence of bacterial cytochromes P-450 that resemble hepatic cytochromes.

Quantitative role of parenchymal and non-parenchymal liver cells in the uptake of [14C]sucrose-labelled low-density lipoprotein in vivo.

In order to assess the relative importance of the receptor for low-density lipoprotein (LDL) (apo-B,E receptor) in the various liver cell types for the catabolism of lipoproteins in vivo, human LDL was labelled with [14C]sucrose. Up to 4.5h after intravenous injection, [14C]sucrose becomes associated with liver almost linearly with time. During this time the liver is responsible for 70-80% of the removal of LDL from blood. A comparison of the uptake of [14C]sucrose-labelled LDL and reductive-methylated [14C]sucrose-labelled LDL ([14C]sucrose-labelled Me-LDL) by the liver shows that methylation leads to a 65% decrease of the LDL uptake. This indicated that 65% of the LDL uptake by liver is mediated by a specific apo-B,E receptor. Parenchymal and non-parenchymal liver cells were isolated at various times after intravenous injection of [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL. Non-parenchymal liver cells accumulate at least 60 times as much [14C]sucrose-labelled LDL than do parenchymal cells accumulate at least 60 times as much [14C]sucrose-labelled LDL than do parenchymal cells when expressed per mg of cell protein. This factor is independent of the time after injection of LDL. Taking into account the relative protein contribution of the various liver cell types to the total liver, it can be calculated that non-parenchymal cells are responsible for 71% of the total liver uptake of [14C]sucrose-labelled LDL. A comparison of the cellular uptake of [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL after 4.5h circulation indicates that 79% of the uptake of LDL by non-parenchymal cells is receptor-dependent. With parenchymal cells no significant difference in uptake between [14C]sucrose-labelled LDL and [14C]sucrose-labelled Me-LDL was found. A further separation of the nonparenchymal cells into Kupffer and endothelial cells by centrifugal elutriation shows that within the non-parenchymal-cell preparation solely the Kupffer cells are responsible for the receptor-dependent uptake of LDL. It is concluded that in rats the Kupffer cell is the main cell type responsible for the receptor-dependent catabolism of lipoproteins containing only apolipoprotein B.

Uptake of free hemoglobin by rat liver parenchymal cells

Parenchymal cells isolated from rat liver are capable of taking up free hemoglobin. Uptake was saturable with a concentration for half maximal velocity of 1.35 mg/ml (1.99 × 10 −5 M) hemoglobin. At a concentration of 0.088 mg/ml, the endocytic index for hemoglobin uptake was 4.5 μl/h per mg of cell protein. This may be compared with the rate of fluid pinocytosis by these cells of 0.025 μl/h per mg of cell protein (determined with yeast invertase as the marker). Free β globin chains were also taken up with an endocytic index of 26.7 μl/h per mg of cell protein at a β chain concentration of 0.075 mg/ml. Hemoglobin inhibited the uptake of labeled β globin. Hemoglobin-haptoglobin complex at a concentration of 0.12 mg/ml (as hemoglobin) was cleared at a rate of 0.89 μl/h per mg cell protein and its uptake was also inhibited by free hemoglobin. We conclude that haptoglobin serves to conserve the iron of hemoglobin by preventing its renal clearance and not by promoting its hepatic uptake.

Changes in the binding of "fast"-form .alpha.2-macroglobulin to 3T3-L1 cells after differentiation to adipocytes

Human alpha 2-macroglobulin (alpha 2M)-CH3NH2 specifically binds to 3T3-L1 fibroblasts and adipocytes with an apparent Kd of 0.3 nM at 4 degrees C. Binding to fibroblasts follows first-order kinetics only for the first 20-30 min of reaction, k1 = 160 microM-1 h-1, and then proceeds in a non-first-order reaction that takes 28 h to reach steady state. Receptor activity is 120 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is nondissociable. In contrast, binding to adipocytes follows first-order kinetics, k1 = 720 microM-1 h-1, and reaches steady state in 6-8 h. Receptor activity is 35 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is reversible with a k2 of 0.4 h-1. Control studies with 3T3-C2 cells, which do not differentiate after hormone treatment, indicate that these differences are not due to hormone treatment alone. Binding to both fibroblasts and adipocytes is specific for "fast"-form alpha 2M but not for native alpha 2M. Inhibition studies with neoglycoproteins demonstrate that binding does not occur via any of the known carbohydrate receptors. Some cross-reactivity with antithrombin III-trypsin complexes is demonstrated. Both fibroblasts and adipocytes take up and degrade alpha 2M-CH3NH2 at 37 degrees C. For both cell types, the concentration of alpha 2M-CH3NH2 needed for half-maximal uptake is 65 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells.

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.

Influence of Environmental Factors and Medium Composition on Vibrio gazogenes Growth and Prodigiosin Production

Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h and a maximum prodigiosin concentration of 80 ng of prodigiosin . mg of cell protein were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h), and maximum prodigiosin concentrations were higher (1,500 ng . mg of protein). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH(2)PO(4) above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion . liter, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng . mg protein) was observed in chemostat cultures at a dilution rate of 0.057 h. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures.

Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum

Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo.

Processing of acetylated human low-density lipoprotein by parenchymal and non-parenchymal liver cells. Involvement of calmodulin?

1. Modified lipoproteins have been implicated to play a significant role in the pathogenesis of atherosclerosis. In view of this we studied the fate and mechanism of uptake in vivo of acetylated human low-density lipoprotein (acetyl-LDL). Injected intravenously into rats, acetyl-LDL is rapidly cleared from the blood. At 10min after intravenous injection, 83% of the injected dose is recovered in liver. Separation of the liver into a parenchymal and non-parenchymal cell fraction indicates that the non-parenchymal cells contain a 30-50-fold higher amount of radioactivity per mg of cell protein than the parenchymal cells. 2. When incubated in vitro, freshly isolated non-parenchymal cells show a cell-association of acetyl-LDL that is 13-fold higher per mg of cell protein than with parenchymal cells, and the degradation of acetyl-LDL is 50-fold higher. The degradation of acetyl-LDL by both cell types is blocked by chloroquine (10-50mum) and NH(4)Cl (10mm), indicating that it occurs in the lysosomes. Competition experiments indicate the presence of a specific acetyl-LDL receptor and degradation pathway, which is different from that for native LDL. 3. Degradation of acetyl-LDL by non-parenchymal cells is completely blocked by trifluoperazine, penfluridol and chlorpromazine with a relative effectivity that corresponds to their effectivity as calmodulin inhibitors. The high-affinity degradation of human LDL is also blocked by trifluoperazine (100mum). The inhibition of the processing of acetyl-LDL occurs at a site after the binding-internalization process and before intralysosomal degradation. It is suggested that calmodulin, or a target with a similar sensitivity to calmodulin inhibitors, is involved in the transport of the endocytosed acetyl-LDL to or into the lysosomes. 4. It is concluded that the liver, and in particular non-parenchymal liver cells, are in vivo the major site for acetyl-LDL uptake. This efficient uptake and degradation mechanism for acetyl-LDL in the liver might form in vivo the major protection system against the potential pathogenic action of modified lipoproteins.

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.

Oxidation-reduction potentials of respiratory chain components in ThiobacillusA2

(1) Cells of Thiobacillus A 2 grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a 3-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 ( E m,7.0) and spectral maxima of c-, b-, a- and a 3-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the ‘invisible’, or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a 3 complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.