Immobilization of the surfactant-degrading bacterium Pseudomonas C12B in polyacrylamide gel beads: I. Effect of immobilization on the primary and ultimate biodegradation of SDS, and redistribution of bacteria within beads during use.

The surfactant-degrading bacterium Pseudomonas C12B was immobilized in polyacrylamide gel beads. Conditions were established for minimizing the apparent loss of sodium dodecyl sulfate (SDS)-degrading activity accompanying polymerization, while still retaining durable gel beads. Apparent losses in SDS-degrading activity compared with free untreated bacteria were attributed largely to substrate diffusion limitations imposed by the gel matrix. Changes in the rate and extent of conversion of radiolabel from [1-14C]SDS to 14CO2 were attributed to diffusional restrictions on O2 availability within the gel beads. Scanning electron microscopy was used to show that beads (3 mm3) repeatedly exposed to SDS for 35 days contained a high cell density in a sub-surface layer 0.4-0.7 mm deep, with relatively few bacteria either at greater depth or at the bead surface.     

In vivo protein synthesis from 14C-substrates in porcine tissues during the transition to postnatal development

[2-14C] leucine, [1-14C] alanine, [1-14C] glucose, [1-14C] lactate and [1-14C] pyruvate utilization in the protein synthesis has been studied in vivo at early stages of postnatal development of piglets. It has been established, that during the first 24 hours after birth the protein synthesis intensity, judging by [2-14C] leucine incorporation, in liver, skeletal muscle, duodenal wall and subcutaneous tissue of piglets increases 5, 7, 6.5 and 2.1 times respectively. At the age of 1-2 h the radioactive carbon incorporation from [1-14C] glucose into the brain proteins is more pronounced than into the proteins of liver and skeletal muscle. During the first days of life the intensity of the label incorporation from [1-14C] glucose into liver and skeletal muscle proteins of piglets is enhanced, whereas in brain it remains at the same level. The degree of 14C carbon incorporation from [1-14C]-alanine, [1-14C] pyruvate and [1-14C] lactate into the liver and skeletal muscle proteins of 5-days-old piglets is approximately the same, 14C substrates of protein synthesis in brain and subcutaneous adipose tissue having some peculiarities.     

A mevalonate requirement for maintenance of fatty acid and protein synthesis during hormonally stimulated development of mammary gland in vitro

The effect of compactin on hormonally induced lipogenesis and protein synthesis was studied in vitro in explants of mammary gland from mid-pregnant rabbits. Compactin blocks mevalonate synthesis by the specific inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, and in this system, culture with 10 microM compactin for 24, 48, and 72 h inhibited incorporation of [1-14C]acetate (but not [2-14C]mevalonate) into sterol by 98, 95, and 86%, respectively. Removal of compactin prior to assay rapidly reversed this effect and was associated with increased tissue 3-hydroxy-3-methylglutaryl-CoA reductase activity. Fatty acid synthesis (measured by incorporation of [1-14C]acetate or [4,5-3H]leucine) and protein synthesis (measured by incorporation of [4,5-3H]leucine) were both inhibited by around 50% after culture with compactin. This inhibition was not rapidly reversed by removal of compactin prior to assay, but it was prevented by inclusion of 1 mM mevalonolactone in the culture medium. After removal of compactin and continued culture in its absence for 24 h with hormones, the normal tissue capacity for fatty acid and protein synthesis was restored, indicating no permanent cell damage. The results suggest a specific requirement for mevalonate (or derived products) for the hormonal maintenance of the increased fatty acid and protein synthesis characteristic of the development of the mammary gland.     

Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin

Isolated Xenopus laevis retinas were incubated with 3H-labeled mannose or leucine in the presence or absence of tunicamycin (TM), a selective inhibitor of dolichyl phosphate-dependent protein glycosylation. At a TM concentration of 20 micrograms/ml, the incorporation of [3H]mannose and [3H]leucine into retinal macromolecules was inhibited by approximately 66 and 12-16%, respectively, relative to controls. Cellular uptake of the radiolabeled substrates was not inhibited at this TM concentration. Polyacrylamide gel electrophoresis revealed that TM had little effect on the incorporation of [3H]leucine into the proteins of whole retinas and that labeling of proteins (especially opsin) in isolated rod outer segment (ROS) membranes was negligible. The incorporation of [3H]mannose into proteins of whole retinas and ROS membranes was nearly abolished in the presence of TM. Autoradiograms of control retinas incubated with either [3H]mannose or [3H]leucine exhibited a discrete concentration of silver grains over ROS basal disc membranes. In TM-treated retinas, the extracellular space between rod inner and outer segments was dilated and filled with numerous heterogeneously size vesicles, which were labeled with [3H]leucine but not with [3H]mannose. ROS disc membranes per se were not labeled in the TM-treated retinas. Quantitative light microscopic autoradiography of retinas pulse-labeled with [3H]leucine showed no differences in labeling of rod cellular compartments in the presence or absence of TM as a function of increasing chase time. These results demonstrate that TM can block retinal protein glycosylation and normal disc membrane assembly under conditions where synthesis and intracellular transport of rod cell proteins (e.g., opsin) are not inhibited.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Acute Effect of Ammonia on Branched-Chain Amino Acid Oxidation and Incorporation into Proteins in Astrocytes and in Neurons in Primary Cultures

14CO2 production and incorporation of label into proteins from the labeled branched-chain amino acids, leucine, valine, and isoleucine, were determined in primary cultures of neurons and of undifferentiated and differentiated astrocytes from mouse cerebral cortex in the absence and presence of 3 mM ammonium chloride. Production of 14CO2 from [1-14C]leucine and [1-14C]valine was larger than 14CO2 production from [U-14C]leucine and [U-14C]valine in both astrocytes and neurons. In most cases more 14CO2 was produced in astrocytes than in neurons. Incorporation of labeled branched-chain amino acids into proteins varied with the cell type and with the amino acid. Addition of 3 mM ammonium chloride greatly suppressed 14CO2 production from [1-14C]-labeled branched chain amino acids but had little effect on 14CO2 production from [U-14C]-labeled branched-chain amino acids in astrocytes. Ammonium ion, at this concentration, suppressed the incorporation of label from all three branched-chain amino acids into proteins of astrocytes. In contrast, ammonium ion had very little effect on the metabolism (oxidation and incorporation into proteins) of these amino acids in neurons. The possible implications of these findings are discussed, especially regarding whether they signify variations in metabolic fluxes and/or in magnitudes of precursor pools.     

Evidence for the glycoprotein nature of retina glycogen

Incubation of a bovine retina membrane preparation with micromolar amounts of UDP-[14C]glucose resulted in the incorporation of [14C]glucose into endogenous (1----4)-alpha-glucan, insoluble in trichloroacetic acid, and acid-soluble ethanol-insoluble glycogen. The trichloroacetic-acid-insoluble glucan fraction of retina migrated in 2.6-3% acrylamide gels when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and was rendered acid-soluble by digestion with pronase. The solubility of the acid-insoluble glucan in acidified organic solvent was different from that of amylose or glycogen and similar to membrane proteins and glycoproteins. The glycogen fraction of retina contained 1.5-2.0 micrograms protein/100 micrograms glucose. When this fraction was analyzed by SDS-PAGE only one band, which moved near the top of 3% acrylamide gels, was stained with periodic acid Schiff reagent and Coomassie blue. The protein nature of the Coomassie-blue-stainable material was demonstrated by iodination of the glycogen fraction with [131I]iodide and identification of labeled monoiodotyrosine and diiodotyrosine. The bulk of the label comigrated with carbohydrate near the top of gels in SDS-PAGE and treatment with alpha- amylse decreased the molecular size of both labeled and stainable material. Physical dissociative conditions (7.5 M urea/0.83% SDS/0.83% mercaptoethanol) and the following chemical treatments failed to dissociate the iodinated protein from glycogen: (a) 0.1 M NaOH/0.1 M NaBH4 at room temperature for 24 h; (b) 1 M HCl in methanol at 50 degrees C for 10 min; (c) trifluoroacetic acid at 50 degrees C for 6 min. 131I-labeled glycogenpeptide was isolated after 131I-labeled protein-bound glycogen had been subjected to digestion with papain/pronase and passed through a Sepharose column. The results suggest that at least part of glycogen in bovine retina is firmly combined to protein as a single proteoglycogen molecule. Furthermore some of the proteoglycogen might be present as a trichloroacetic-acid-precipitable proteoglucan owing to its lower glucose content.     

Induction of cyst coat protein synthesis by starvation in the ciliate Colpoda steinii

1. At 28 degrees C, synthesis of protein cyst coat in ciliates of Colpoda steinii is induced by washing with water and, as judged by glutamic acid assays and incorporation studies with l-[U-(14)C]leucine, starts about 30min after the cells have stopped swimming and is largely complete 90min later. During this time up to 70% of the protein synthesized by the cell is coat protein. 2. When cells were placed in l-[U-(14)C]leucine at low concentrations (0.25-0.76mm) during the period of coat synthesis there was no lag in uptake. Only a small proportion of the leucine incorporated into the coat was from the external substrate, implying that the rate of radioactive isotope incorporation measured the rate of transport of amino acid into the cell. Transport of l-[U-(14)C]leucine into the cell was markedly stimulated by l-glutamic acid and l-lysine. 3. When cells were placed in l-[U-(14)C]leucine at high concentrations (38mm) the rate of incorporation was considered to measure the rate of protein synthesis, but because the latter may have been affected by substrate it is concluded that such measurements are of doubtful value.     

Phycobiliprotein synthesis in protoplasts of the unicellular cyanophyte, Anacystis nidulans.

Stable and metabolically active protoplasts were prepared from the unicellular cyanophyte, Anacystis nidulans, by enzymatic digestion of the cell wall with 0.1% lysozyme. The yield of protoplasts from intact algal cells was approx. 50%. Incorporation of L-[U-14C]leucine into cold trichloroacetic acid-insoluble material from protoplasts preparations was linear for 1.5 h and continued for an additional 2.5 h. Incorporation of radiolabeled leucine into hot trichloroacetic acid-insoluble material from protoplast preparations demonstrated protein synthesis in protoplasts in vitro. Phycocyanin is the principal phycobiliprotein and allophycocyanin is a minor phycobiliprotein in A. nidulans cells. The light-absorbing chromophore of both of these phycobiliproteins is the linear tetrapyrrole (bile pigment), phycocyanobilin. Radiolabeled phycocyanin and allophycocyanin were isolated from protoplast preparations which had been incubated with L-[U-14]leucine or delta-amino[4-14C] levulinic acid (a precursor of phycocyanobilin). The radio-labeled phycobiliproteins were purified by ammonium sulfate fractionation and ion-exchange chromatography on brushite columns. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled leucine) was 106 000 and 82 000 dpm/mg, respectively. The specific radioactivity of phycocyanin and allophycocyanin in brushite column eluates (protoplasts incubated with radiolabeled delta-aminolevulinic acid) was 33 000 and 38 000 dpm/mg, respectively. Phycobiliproteins from protoplasts incubated with radiolabeled leucine were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 25% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated radioactivity in protoplast lysates and approx. 60% of the incorporated ratioactivity in phycocyanin and allophycocyanin (in brushite column eluates) comigrated with the subunits of these phycobiliproteins on sodium dodecyl sulfate-polyacrylamide gels. Chromic acid degradation of phycobiliproteins from protoplast preparations incubated with delta-amino[4-14C] levulinic acid yielded radiolabeled imides which were derived from the phycocyanobilin chromophore. Imides from radiolabeled phycobiliproteins isolated from protoplast preparations incubated with L-[U-14C]leucine did not contain radioactivity. These results show that both the apoprotein and tetrapyrrolic moieties of phycocyanin and allophycocyanin were synthesized in A. nidulans protoplasts in vitro.     

Rhamnogalacturonan-II--a biologically active fragment

Rhamnogalacturonan-II inhibited the uptake of [¹⁴C]leucine and,consequently, the incorporation of [¹⁴C]leucine into acid-precipitableproteins by suspension-cultured tomato cells. Fractionationof rhamnogalacturonan-II showed that the lower molecular componentswere the most effective. KDO and apiose, both constituents ofrhamnogalacturonan-II, also inhibited [¹⁴C]leucine incorporationweakly, suggesting that these sugar residues may be an integralrequirement for the biological activity of rhamnogalacturonan-II.The incorporation of [¹⁴C]glutamate and [¹⁴C]histidine, andto a lesser extent [¹⁴C]proline and [¹⁴C]arginine, was alsoinhibited by rhamnogalacturonan-II; the incorporation of [¹⁴C]tyrosineand [¹⁴C]phenylalanine was little affected. This suggests thatrhamnogalacturonan-II exerts its effect by acting on certainmembrane transport systems. Key words: Rhamnogalacturonan-II, inhibition, protein synthesis, amino acid incorporation     

Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro.

1. The turnover rate of L-[1-14C]leucine was increased by 35% in lactating rats compared with virgin rats. Starvation or removal of pups (24 h) returned the value to that of the virgin rat. 2. Incorporation of L-[U-14C]leucine into lipid and protein of mammary glands of lactating rats in vivo increased 7-fold and 6-fold respectively compared with glands of virgin rats. Lactation caused no change in the incorporation of L-[U-14C]leucine into hepatic lipid and protein. 3. The production of 14CO2 from L[l-14C]leucine (in the presence of glucose) was similar in isolated acini from glands of fed (chow) and starved lactating rats. Feeding with a 'cafeteria' diet caused a slight decrease, and removal of pups a large decrease, in the oxidative decarboxylation of leucine. 4. Oxidation of L-[2-14C]leucine to 14CO2 was increased about 3-fold in acini from starved lactating rats or lactating rats fed on a 'cafeteria' diet compared with rats fed on a chow diet. Insulin decreased the formation of 14CO2 in all three situations. 5. Incorporation of L-[U-14C]- and [2-14C]-leucine into lipid was decreased in acini from starved lactating rats and lactating rats fed on a 'cafeteria' diet. Insulin tended to increase the conversion of [2-14C]leucine into lipid, but this was significant only in the case of the acini from 'cafeteria'-fed rats. 6. Experiments with (-)-hydroxycitrate indicate that the major route for conversion of leucine carbon into lipid in acini is via citrate translocation from the mitochondria. 7. The physiological implications of these findings are discussed.     

Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

Characterization of kappa-carrageenan gels used for immobilization of Bacillus firmus.

In this study, aimed at a biochemical and physical characterization of kappa-carrageenan gels used for entrapment of Bacillus firmus NRS 783 (a superior producer of an alkaline protease), effects of carrageenan concentration, gelation temperature, initial cell loading, and strength of the curing agent (KCl) on the properties of cell-free and cell-laden gels were examined. The physical properties of the differently prepared gels that were examined included density, free volume fraction, mechanical strength, and change in gel volume during gel curing. The biochemical characteristics studied included viability of gel-entrapped cells, cell leakage from cell-laden gels, and cell penetration into cell-free gels. For the range of carrageenan contents investigated [between 2% and 5% (w/v)], the mechanical strength of the gels with/without KCl curing was observed to increase with an increase in carrageenan content of gels. The mechanical strength of each gel increased substantially upon extensive curing. Free volume fractions in excess of 0.8 were observed for all gels. Of cells that were viable prior to immobilization, 90-92% remained viable after formation and extensive curing of gels for cell-gel mixtures prepared at 45 degrees C. Attempts at prolonged storage of cell-laden gel beads at 0 degrees C as stock cultures of immobilized B. firmus were unsuccessful due to a significant decline in cell viability during such storage. On the basis of the cell leakage studies, the average pore sizes of 2%, 3%, 4%, and 5% gels were deduced to increase in the following order of carrageenan content (w/v): 4%, 3%, 2%, and 5%. Commensurate with the decrease in the average pore size (or the increased tightness of the gels) with increasing carrageenan content, both the extent of cell leakage and the extent of net cell penetration decreased with increasing carrageenan content for the first three gels. Owing to non-uniform distribution of free space and much larger pores, the extent of net cell penetration in 5% carrageenan gels was considerably low, while the extent of cell leakage in 5% carrageenan gels was an order of magnitude greater than the extents of cell leakage in the other three gels.     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Characterization of the cell wall and outer membrane of Rhodopseudomonas capsulata.

Sucrose density gradient centrifugation of cell envelopes of chemotrophically grown cells of Rhodopseudomonas capsulata St. Louis (= ATCC 23782) resulted in the separation of a cytoplasmic membrane from a cell wall fraction (buoyant densities, 1.139 and 1.215 g/cm3, respectively). The cell wall fractions (untreated or Triton extracted) contained peptidoglycan- and lipopolysaccharide-specific components. Their neutral sugar content, mainly rhamnose and galactose, was high (250 and 100 micrograms/mg [dry weight] of material) due to a non-lipopolysaccharide polymer. The fatty acid content was low (less than or equal to 60 micrograms/mg [dry weight] of material), and half of it was contributed by lipopolysaccharide (3-OH-C10:0, C12:1, and 3-oxo-C14:0). The predominant other fatty acid was C18:1. An outer membrane fraction, obtained by lysozyme treatment of the Triton-extracted cell wall, showed essentially the same chemical composition except for almost complete removal of peptidoglycan. Saline extraction (0.9% NaCl, 37 degrees C, 2 h) removed a lipopolysaccharide-protein(-phospholipid?) complex from whole cells of R. capsulata St. Louis. The polypeptide patterns of the cell wall and outer membrane as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comprised 20 to 25 different polypeptides (most of them very faint) and were dominated by a single, heat-modifiable major protein (Mr 69,000 after solubilization below 60 degrees C; Mr 33,000 at temperatures above 70 degrees C).     

Stimulation of rat uterine collagen synthesis by relaxin

Immature, ovariectomized, estrogen-primed rats respond to the administration of porcine relaxin by an increase in the incorporation of labeled amino acids ([14C]leucine, [14C]phenylalanine, [3H]proline) into uterine proteins in vitro. The maximum response occurs about 12 hr after a single injection of 0.1 mg relaxin in benzopurpurine 4B solution; subsequently, the relaxin effect declines but is still apparent after 24 hr. Smaller, but still significant increases in incorporation rates can be induced by relaxin in the absence of estrogen priming. Uterine collagen synthesis, as indicated by the incorporation of [3H]proline and its conversion to hydroxyproline, appears to be a primary target of the relaxin stimulus, since the effect of relaxin upon proline incorporation into uterine collagen is significantly greater than its effect upon labeling of noncollagen protein.     

Synthesis and turnover of the regularly arranged surface protein of Acinetobacter sp. relative to the other components of the cell envelope.

The formation of the components of the cell envelope of Acinetobacter sp. 199A was investigated by measuring the incorporation of [3H]leucine into protein, [14C]galactose into lipopolysaccharide, 32P into phospholipid, and [3H]diaminopimelic acid into peptidoglycan. Whereas the lipopolysaccharide and intrinsic protein of the outer membrane were stable, some of the regularly arranged surface protein, the alpha-protein, was lost into the growth medium. Only newly synthesized alpha-protein was lost. The peptidoglycan of the murein layer was also labile. Selective inhibition of the formation of individual components of the cell envelope with penicillin, chloramphenicol, and bacitracin showed that incorporation of protein into the outer membrane required the simultaneous formation of complete lipopolysaccharide. The converse was not true: protein synthesis was not required for lipopolysaccharide incorporation. Formation of the outer membrane and the murein layer proceeded independently.     

Induction of specific proteins in cultured skeletal muscle cells by 1,25-dihydroxyvitamin D-3

The presence in myoblasts of an intracellular receptor specific for 1,25-dihydroxyvitamin D-3 [1,25(OH)2D3) and 1,25(OH)2D3-dependent changes in myoblast Ca2+ transport and phospholipid metabolism which are suppressed by RNA and protein synthesis inhibitors have been shown. In agreement with these observations, incubation of chick embryo myoblasts, precultured for 24 h in a medium containing low levels of vitamin D-3 metabolites, with 1,25(OH)2D3 at conditions which induce maximum cell responses (10(-10) M, 24 h) markedly stimulated the incorporation of [3H]leucine into total cell proteins and this effect was abolished when sterol treatment was performed in the presence of cycloheximide or puromycin. To investigate whether 1,25(OH)2D3 selectively stimulates the de novo synthesis of muscle cell proteins, mixtures of myoblast proteins from control and sterol-treated cultures labelled with [14C]leucine and [3H]leucine, respectively, were separated by SDS-polyacrylamide gel electrophoresis and isoelectric focussing. Examination of 3H/14C ratios in gel fractions revealed that 1,25-(OH)2D3 stimulates the production of proteins of molecular masses (isoelectric points) of 9 kDa (4.1 and 8.5), 17 kDa (7.5), 30 kDa (7.2), 40 kDa (5.5), 55 kDa (4.5) and 100 kDa (8.6). Cell fractionation studies showed the following subcellular distribution: 9 kDa (85% cytosol, 15% microsomes); 17 and 100 kDa (100%, 1200 X g pellet); 30 kDa (65% cytosol, 35% mitochondria); 40 kDa (100% microsomes); 55 kDa (65% microsomes, 35% mitochondria). Marker enzyme data indicated that this distribution is not due to cross-contamination between fractions. Affinity chromatography of double-labelled myoblast proteins on an immobilized lectin showed that the 55 kDa protein contains carbohydrate. Labelling of myoblast proteins with 45CaCl2 after their separation on SDS-polyacrylamide gels showed in addition that the 1,25(OH)2D3-dependent proteins of 9, 17, 40 and 100 kDa are major Ca2+-binding components of the cells. Synthesis of these proteins may mediate the effects of the sterol on myoblast calcium metabolism.     

Comparison of the effect of acetone and isopropanol on lipid metabolism in rats]

Isopropanol and acetone administered to rats in conditions leading to a similar blood acetone level differ markedly in their effects on lipid metabolism. Isopropanol administration determines a fatty liver, which is mainly related to a defect in hepatic lipoprotein synthesis. Acetone administration gives only raise to a slight increase in the liver triacylglycerol level. It does not alter the [1-14C] palmitate, [1-14C] glycerol or [U-14C] leucine incorporation into blood lipoproteins. Acetone does thus not appear to play a preminent role in the isopropanol induced fatty liver which seems to be related mainly to a direct action of the alcohol itself.