Use of lignin-degrading fungi in the disposal of pentachlorophenol-treated wood

Summary The lignin-degrading fungiPhanerochaete chrysosporium, P. sordida, Trametes hirsuta, andCeriporiopsis subvermispora were evaluated for their ability to decrease the concentration of pentachlorophenol (PCP) and to cause dry weight loss in PCP-treated wood. Hardwood and softwood materials from PCP-treated ammunition boxes that were chipped to pass a 3.8-cm screen were used. All four fungi caused significant weight losses and decreases in the PCP concentration. The largest PCP decrease (84% in 4 weeks) was caused byT. hirsuta, and the smallest decrease was caused byC. subvermispora (37% in 4 weeks). After 4 weeks, the fate of spiked¹⁴C[PCP] in softwood chips inoculated withT. hirsuta was as follows: 27% was mineralized, 42.5% was non-extractable and bound to the chips, 23.5% was associated with fungal hyphae, and 6% was organic-extractable. Decreases of PCP byP. chrysosporium andP. sordida averaged 59% and 57%, respectively. PCP decreases caused byPhanerochaete spp. were not significantly affected by wood type or sterilization and were primarily due to methylation of PCP that resulted in accumulation of pentachloroanisole. Softwood weight losses caused byT. hirsuta, P. chrysosporium andC. subvermispora were respectively, 24, 6.5, and 17%, after 4 weeks. These weight losses are comparable to reported weight losses by these organisms in non-treated softwood. Nutrient supplementation significantly increased weight loss but not percentage decrease of PCP. The results of this research demonstrate the potential for using lignin-degrading fungi to destroy PCP-treated wood.     

CD4+-T-Cell and CD20+-B-Cell Changes Predict Rapid Disease Progression after Simian-Human Immunodeficiency Virus Infection in Macaques

Simian-human immunodeficiency virus 89.6PD (SHIV_89.6PD) was pathogenic after intrarectal inoculation of rhesus macaques. Infection was achieved with a minimum of 2,500 tissue culture infectious doses of cell-free virus stock, and there was no evidence for transient viremia in animals receiving subinfectious doses by the intrarectal route. Some animals experienced rapid progression of disease characterized by loss of greater than 90% of circulating CD4⁺ T cells, sustained decreases in CD20⁺ B cells, failure to elicit virus-binding antibodies in plasma, and high levels of antigenemia. Slower-progressing animals had moderate but varying losses of CD4⁺ T cells; showed increases in circulating CD20⁺ B cells; mounted vigorous responses to antibodies in plasma, including neutralizing antibodies; and had low or undetectable levels of antigenemia. Rapid progression led to death within 30 weeks after intrarectal inoculation. Plasma antigenemia at 2 weeks after inoculation (P ≤ 0.002), B- and T-cell losses (P ≤ 0.013), and failure to seroconvert (P ≤ 0.005) were correlated statistically with rapid progression. Correlations were evident by 2 to 4 weeks after intrarectal SHIV inoculation, indicating that early events in the host-pathogen interaction determined the clinical outcome.     

Modelling the relationship between purple blotch and yield loss in garlic and effect of leaf damage on bulb yield

An AUDPC model was developed to describe the relationship between Alternaria porri infection and yield loss in garlic. Percentage yield loss was regressed against AUDPC and gave an acceptable fit to a linear model with an intercept of -0.35, a slope of 0.09 and R²= 0.85 during 1990–91 and an intercept of 1.91, a slope of 0.08 and R²= 0.87 during 1991–92. The effect of various levels of leaf damage on garlic bulb yield was also studied to simulate damage caused by A. porri. Significant yield reduction by 25% defoliation at 5 wk, 50% defoliation at 4 wk and 75% defoliation at 3 wk before bulb maturity were recorded. No significant yield reduction from 2 wk before bulb maturity could, however, be noticed even at the highest levels of leaf damage. Linear regression models were fitted for predicting yield at various levels of defoliation at different weeks before bulb maturity. These models can be used to describe the consequences of disease epidemics, projecting possible losses during the season and justifying the need for chemical disease control.     

Uptake and degdradation of formaldehyde-treated 125I-labeled human serum albumin in rat liver cells in vivo and in vitro

The uptake of formaldehyde-treated ¹²⁵I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20–30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.     

Bovine oocytes in secondary follicles grow and acquire meiotic competence in severe combined immunodeficient mice

Cortical tissues containing only primordial and primary follicles, or secondary follicles 140-190 microm in diameter, were collected from bovine ovaries and xenografted under the kidney capsules of female severe combined immunodeficient (SCID) mice. Histological examination revealed that all grafts were well vascularised and contained surviving follicles at 4 or 6 weeks after grafting. Primordial and primary follicles survived but did not develop beyond the one-layer stage. Secondary follicles, on the other hand, had formed antra at 4 weeks after grafting. The mean diameter of secondary follicles, which was 165.2 +/- 17.0 microm (n = 42) before grafting, had developed to 442.9 +/- 77.9 microm (n = 37) and 592.9 +/- 116.0 microm (n = 45) in diameter at 4 and 6 weeks after grafting, respectively. The mean diameter of oocytes, which was 55.1 +/- 4.9 microm (n = 42) before grafting, also increased significantly (4 weeks: 105.6 +/- 6.3 microm; 6 weeks: 122.2 +/- 2.6 microm; p < 0.05). Oocytes were recovered from follicles that had developed to more than 400 microm in diameter after 6 weeks, and were subjected to subsequent mature culture. Of these oocytes, 34% (11/32) resumed meiosis and 6% (2/32) matured to the second metaphase. Follicular fluid in bovine antral follicles developed in SCID mice had the 69 kDa protein, which was detected by anti-mouse albumin antibody but not by anti-bovine albumin antibody in immunoblotting analysis. These results demonstrated that bovine secondary follicles develop to the antral stage in SCID mice, and that the oocytes in the follicles acquire the meiotic competence.     

Vascular and epithelial damage in the lung of the mouse after X rays or neutrons

The response of the lung was studied in CFLP mice after exposure of the whole thorax to X rays (250 kVp) or cyclotron neutrons (16 MeV deuterons on Be, mean energy 7.5 MeV). To measure blood volume and leakage of plasma proteins, 51Cr-labeled red blood cells and 125I-albumin were injected intravenously and 24 h later lungs were lavaged via the trachea. Radioactivities in lung tissue and lavage fluid were determined to estimate the accumulation of albumin in the interstitial and alveolar spaces indicating damage to blood vessels and alveolar epithelium respectively. Function of type II pneumonocytes was assessed by the amounts of surfactant (assayed as lipid phosphorous) released into the lavage fluid. During the first 6 weeks, lavage protein and surfactant were increased, the neutron relative biological effectiveness (RBE) being unity. During pneumonitis at 12-24 weeks, surfactant levels were normal, blood volume was decreased, and both interstitial and alveolar albumin were increased. Albumin levels then decreased. At late times after exposure (42-64 weeks) alveolar albumin returned to normal but interstitial albumin was still slightly elevated. Values of RBE for changes in blood volume and interstitial and alveolar albumin at 15 weeks and for changes in blood volume and interstitial albumin at 46 weeks were 1.4, comparable with that for animal survival at 180 days. The results indicate that surfactant production is not critical for animal survival. They suggest that changes in blood vessels and alveolar epithelium occur during acute pneumonitis; epithelial repair follows but some vascular damage may persist. The time course of the changes in albumin levels did not correlate with increases in collagen biosynthesis which have been observed as early as 1 month after exposure and persist for up to 1 year. Furthermore, a dose which had no effect on leakage caused a marked increase in collagen biosynthesis. Thus the present results do not support a causal relationship between exudation of vascular protein during pneumonitis and the later development of fibrosis.     

Labeling and tracking human amniotic epithelial cells with green fluorescent protein in an adeno-associated virus vector

Human amniotic epithelial cells (hAECs) are a recently identified type of stem cell. Thanks to their ready availability and the lower risk of teratoma formation, hAECs have been studied and tested for a variety of human disease treatments and tissue reconstruction efforts. This aim of this study was to establish a stable tracking system to further monitor hAECs in vivo after transplantation. hAECs were isolated from the placentas of patients who visited the Hunan Province Maternity and Child Care Hospitals between Jan 2008 and Jan 2009. Using the classic transfection/infection technique, we successfully introduced green fluorescent protein (GFP) into cultured hAECs with an adeno-associated virus (AAV) vector. The initial preparation of the AAV-GFP virus stock was titrated using HT1081 cells, and further used for the infection of hAECs. GFP⁺ hAECs preserve the capacity of differentiation into hepatocytelike cells with the expression of cytokeratin-18 (CK18) and albumin (ALB). AAV-GFP virus-infected hAECs were transplanted through the spleen into severe combined immune deficiency (SCID) mice via hepatectomy. Four weeks later, the GFP and human albumin expressions were examined in multiple organs through immunoflourence staining. In culture, over 50% of the hAECs were GFP-positive 3 days after infection. Following transplantation, AAV-GFPinfected hAECs survived and continued to express GFP in the host for up to 4 weeks. These cells were primarily found in the spleen and liver, expressing human albumin. This study provides a feasible and stable system to track hAECs. It may prove useful to further identify their biological characteristics after transplantation and to elucidate their beneficial roles for therapeutic purposes.     

Nitrogen losses due to denitrification from cattle slurry injected into grassland soil with and without a nitrification inhibitor

Injection of cattle slurry into a grassland soil decreases NH₃ volatilisation and increases N utilisation by the sward, but may also increase denitrification losses. Denitrification rates were measured using a soil core incubation technique involving acetylene inhibition, following injection of cattle slurry (67 t ha^–1) into a grassland soil. The slurry was injected, either with or without a nitrification inhibitor (DCD), on 8 December 1989. Two-weekly measurements were carried out up to 18 weeks after injection. Compared to the control plot, denitrification rates were significantly higher after slurry injection. Addition of DCD to the slurry almost eliminated this effect. Estimated N-losses during 18 weeks after injection were 0.9 (control), 4.1 (+DCD), and 13.7 (-DCD) kg N ha^–1. Denitrification losses were 7% of the injected NH₄-N and decreased to 2% of the injected NH₄-N when DCD was added. Denitrification could account for about 19% of the difference in apparent recovery of N from slurry injected with and without DCD. The results suggested that considerable amounts of NO₃ ^– were lost due to leaching.     

Andropogon gayanus (Gamba Grass) Invasion Increases Fire-mediated Nitrogen Losses in the Tropical Savannas of Northern Australia

Invasive alien grasses can substantially alter fuel loads and fire regimes which could have significant consequences for fire-mediated nutrient losses. The effects of the alien grass Andropogon gayanus Kunth. (Gamba grass) on fire-mediated nutrient losses was evaluated in Australia’s tropical savannas. Losses of macronutrients during fire were determined by comparing the nutrient pools contained in the fine fuel before fire and in the ash after fire. Pre-fire grass nutrient pools were significantly higher in A. gayanus plots than in native grass plots for all nutrients measured (N, P, K, S, Ca, and Mg). Nutrient losses were substantially higher in A. gayanus plots, with 113% higher losses for N, 80% for P, 56% for K, 63 for S, 355% for Ca, and 345% for Mg. However, only losses of N and Mg varied significantly between grass types. A simplified savanna ecosystem nutrient budget estimated that A. gayanus fires led to the net N loss of 20 kg ha⁻¹ y⁻¹. This is a conservative estimate because total fuel loads were relatively low (7.85 t ha⁻¹) for A. gayanus invaded plots leading to a relatively moderate intensity fire (6,408 kW m⁻¹). Higher A. gayanus fuel loads and fire intensities could potentially lead to losses of up to 61.5 kg N ha⁻¹ from the grass fuel. Over the long term, this is likely to lead to depletion of soil nutrients, particularly N, in the already low-fertility tropical savanna soils.     

High albumin production by multicellular spheroids of adult rat hepatocytes formed in the pores of polyurethane foam

Summary Adult rat hepatocytes formed spherical multicellular aggregates (spheroids) when they were cultured in the pores of polyurethane foam (PUF). The diameter of the spheroids was within the range 100–200 m. These spheroids partly attached and immobilized in the PUF pores for at least 2 weeks. The albumin production rate by the spheroids increased up to 17.0 g/10⁶ nuclei per day during the first 6 days and maintained at a high level for 2 weeks. In contrast, the albumin production rate by the monolayer markedly decreased after 3 days. The spheroid culture using PUF seems to be a convenient and simple method for maintaining some differentiated functions of hepatocytes and for making a bioreactor using the function of spheroids. Offprint requests to: K. Funatsu     

Stimulation of albumin synthesis in mouse hepatoma cells by Bt2cAMP: cell cycle specificity

Addition of 1 mm dibutyryl cyclic AMP (Bt₂cAMP) to cultures of mouse hepatoma cells, Hepa, specifically stimulates the synthesis of serum proteins including albumin. This stimulation is accompanied by an inhibition of cell proliferation. We have investigated these phenomena in synchronous cultures of Hepa. Proliferation of Hepa was arrested by isoleucine starvation. Synchronous growth was initiated by addition of complete growth medium or complete growth medium supplemented with 1 mm Bt₂cAMP. S phase and mitosis were estimated by determinations of [³H]thymidine incorporation and by cell numbers. The rate of albumin synthesis relative to total protein synthesis was measured by pulse labeling cultures for 30 min with [³H]leucine and comparing amounts of immunoprecipitable label with trichloroacetic acid-precipitable label. Treatment of synchronous cultures with Bt₂cAMP did not alter the duration of S phase or the onset of mitosis. The relative rate of albumin synthesis in Bt₂cAMP-treated culture began increasing after mitosis. The timing of the Bt₂cAMP stimulation of albumin synthesis was further investigated by adding Bt₂cAMP to cultures of Hepa at various times after the initiation of synchronous growth. The relative rate of albumin synthesis was then measured at a fixed postmitotic time. An increased relative rate of albumin synthesis was observed only in cultures exposed to Bt₂cAMP before or during S phase. Thus the postmitotic increase in the synthesis of albumin requires the presence of Bt₂cAMP during S phase.     

Losses of14C from roots of pulse-labelled wheat and barley during washing from soil

In crop carbon budget studies losses of root material during storage and washing of samples may cause considerable errors. To correct data from field experiments where rhizosphere C fluxes in wheat and barley were determined by¹⁴C pulse-labelling at different development stages, experiments were performed to quantify losses of¹⁴C from roots during washing. Losses of¹⁴C from wheat roots grown on nutrient solution and stored in different ways, decreased from on average 45% of total¹⁴C content 8 days after labelling to 27% after 21 days. This decrease was probably related to the incorporation of¹⁴C into structural compounds. During washing of oven-dried soil cores of held-grown wheat and barley 3 weeks after labelling, different size classes of losses of¹⁴C from the roots increased substantially with the development stage of the crop at labelling. The 0.3–0.6 mm size class increased from 5% of the¹⁴C in roots > 0.3 mm in young plants to 25% at ripening, and the < 0.3 mm size class increased from 8 to 41% of total¹⁴C content. The latter size class was, however, determined by washing handpicked roots and may therefore partly consist of adhering exudates, mucilages and microorganisms. The effect of development stage on root washing losses was attributed to root senescence which increases the fragility of roots. Thus, especially at the rate development stages root washing losses caused a severe underestimation of the root¹⁴C content. However, with these results the¹⁴C distribution patterns of the field experiments could be adequately corrected.Communication No. 77 of the Dutch Programme on Soil Ecology of Arable Farming Systems.     

Factors controlling nitrification and denitrification: A laboratory study with gel-stabilized liquid cattle manure

Nitrification and denitrification were studied in a millimeterscale microenvironment using a two-phase system with a liquid manure-saturated layer. Samples consisted of liquid cattle manure and air-dried soil stabilized with silica gel, placed between two aerobic soil phases with a water content near field capacity. A high potential for NH₄ ⁺ oxidation developed within 0–2 mm distance from the interface, and NH₄ ⁺ diffused only 10–20 mm into the soil. Some NH₄ ⁺ was probably immobilized by microorganisms in the soil between 0 and 4 days, after which nitrification was the only sink for NH₄ ⁺. A potential for denitrification developed within the manure-saturated zone. Maximum rates of both potential and actual denitrification were recorded by Day 4, but denitrification continued for at least 2–3 weeks. The potential for nitrification peaked after 14 days. When the pH of the manure was adjusted to 5.5, nitrification was reduced close to the interface, and NH₄ ⁺ penetrated further into the soil before it was oxidized. The pH adjustment had an inhibitory effect on denitrification: Both potential and actual rates of denitrification were almost eliminated for several days. The size of the manure-saturated layer strongly affected denitrification losses. With layers of 8 and 16 mm thickness, losses equivalent to 33 and 40% of the original NH₄ ⁺ pool, respectively, were estimated. When manure corresponding to a 12 mm layer was homogeneously mixed with the soil, only 0.3% was lost.Offprint requests to: S. O. Petersen.     

The sequential synthesis of the polypeptide chain of serum albumin by the microsome fraction of rat liver

1. The isolated microsome fraction of regenerating rat liver was incubated with cell sap, a source of energy and [³⁵S]methionine, [¹⁴C]isoleucine or [¹⁴C]leucine for different periods of time, and microsomal albumin isolated. 2. The distribution of these isotopes in albumin was determined by separation of tryptic peptides from the protein. Radioactivity was measured in peptides either qualitatively by radioautography or quantitatively by labelling with both ³H and ¹⁴C. 3. A gradient of radioactivity existed at all times in albumin isolated after incubating microsomes. 4. The shorter the incubation time the fewer the peptides labelled in albumin, but the peptides with highest specific activity after short incubation times corresponded to those with highest specific activities after long incubation times. 5. Leucine released from the C-terminus of albumin had a higher specific activity than the mean specific activity of the remaining leucine residues in albumin. 6. The peptide with the highest specific activity in albumin is probably derived from the C-terminus of the protein. 7. [¹⁴C]Glutamic acid is incorporated into the N-terminus of albumin after incubating the microsome fraction with this isotopically labelled amino acid, cell sap and a source of energy. The specific activity of the N-terminal glutamic acid under these conditions is less than the mean specific activity of the remaining glutamic acid and glutamine residues in albumin. 8. The results are interpreted as reflecting a sequential synthesis of serum albumin in the isolated microsome fraction of rat liver. The direction of synthesis of albumin is from the N-terminus towards the C-terminus. 9. The bulk of incorporation of radioactive amino acid into albumin in the isolated microsome fraction is due to completion of partially completed, pre-existing peptide and polypeptide chains. A limited synthesis of new chains of albumin does, however, occur.     

前列地尔联合川芎嗪治疗早期糖尿病肾病的疗效观察

目的:观察前列地尔(凯时)联合川芎嗪注射液(川青)治疗糖尿病肾病的临床疗效,探讨其降低尿蛋白,减轻肾损害的机制.方法:收集早期2型糖尿病患者120例,随机分成川芎嗪组(40例)、前列地尔组(40例)和联合治疗组(40例).全部病例进行临床观察2周,分别比较三组血肌酐(Cr)、尿素氮(BUN)、24 h尿蛋白定量治疗前后的变化.结果:治疗2周后,联合治疗组降低24 h尿蛋白定量的作用优于川芎嗪组和前列地尔组差异有统计学意义(P＜0.05).前列地尔组和川芎嗪组比较,差异有统计学意义(P＜0.05).结论:静脉应用前列地尔联合川芎嗪注射液能够降低糖尿病肾病患者尿蛋白,延缓糖尿病肾病的进展,值得临床推广.     

Fixation, Counting, and Manipulation of Heterotrophic Nanoflagellates

Quantitative effects of several fixatives on heterotrophic nanoflagellates (HNAN) and phototrophic nanoflagellates (PNAN) were investigated by hemacytometer and epifluorescence counting techniques. Counts of Monas sp. cultures before and after fixation with unbuffered 0.3% glutaraldehyde and 5% formaldehyde showed no loss of cells during fixation, and cell concentrations remained constant for several weeks after fixation. Buffering of fixatives with borax caused severe losses, up to 100% within 2 h. Field samples from Lake Vechten showed no decline of HNAN and total nanoflagellate concentrations for at least 1 week after fixation with 5% formaldehyde and with 1% glutaraldehyde. With 1% glutaraldehyde, the chlorophyll autofluorescence of PNAN was much brighter than with 5% formaldehyde, although it was lost after a few days and thus limited the storage time of samples. However, when primulin-stained slides were prepared soon after fixation and stored at −30°C, the loss of autofluorescence was prevented and PNAN and HNAN concentrations were stable for at least 16 weeks. Effects of filtration and centrifugation on HNAN were also studied. Filtration vacuum could not exceed 3 kPa since 10 kPa already caused losses of 15 to 20%. Similar losses were caused by centrifugation, even at low speed (500 × g).     

The Lack of Selective Reincorporation into Tissue Protein of the Amino Acids Derived from the Catabolism of Serum Protein

Homologous S³⁵-labeled albumin, gamma globulin, and alpha-beta globulin were transfused into rabbits and the specific activities of the electrophoretic fractions of the sera of the recipients were determined at various time intervals up to 12 days after injection. Detectable reincorporation into a fraction other than that transfused was found only in the gamma globulin fraction after albumin injection. This activity rose between 2 and 12 days and reached a level of 2 to 3 per cent of the extrapolated zero time activity of the albumin fraction. When homologous serum protein doubly labeled with I¹³¹ and S³⁵ was transfused into mice, marked drops in the ratios of I¹³¹ to S³⁵ in the serum and tissue proteins were observed between 1 and 48 hours after injection. On the basis of a determination of the absolute and relative amounts of I¹³¹ and S³⁵ found in the various tissue and serum proteins, the amount of reincorporation of S³⁵ into each protein was calculated. The relative amounts of reincorporation of S³⁵ among the various tissues were remarkably similar to the relative amounts of incorporation of S³⁵ after the injection of labeled free amino acids. It is concluded that serum protein does not form a major direct source of amino acids to the tissues but feeds them indirectly through the extracellular pool.     

Metabolite secretion,Fe3+-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869

Trametes versicolor is a promising white-rot fungus for the biological pretreatment of lignocellulosic biomass. In the present work, T. versicolor ATCC 20869 was grown on Pinus taeda wood chips under solid-state fermentation conditions to examine the wood-degrading mechanisms employed by this fungus. Samples that were subjected to fungal pretreatment for one-, two- and four-week periods were investigated. The average mass loss ranged from 5 % to 8 % (m m⁻¹). The polysaccharides were preferentially degraded: hemicellulose and glucan losses reached 13.4 % and 6.9 % (m m⁻¹) after four weeks of cultivation, respectively. Crude enzyme extracts were obtained and assayed using specific substrates and their enzymatic activities were measured. Xylanases were the predominant enzymes, while cellobiohydrolase activities were marginally detected. Endoglucanase activity, β-glucosidase activity, and wood glucan losses increased up to the second week of biodegradation and remained constant after that time. Although no lignin-degrading enzyme activity was detected, the lignin loss reached 7.5 % (m m⁻¹). Soluble oxalic acid was detected in trace quantities. After the first week of biodegradation, the Fe³⁺-reducing activity steadily increased with time, but the activity levels were always lower than those observed in the undecayed wood. The progressive wood polymer degradation appeared related to the secretion of hydrolytic enzymes, as well as to Fe³⁺-reducing activity, which was restored in the cultures after the first week of biodegradation.     

Effects of cyclic AMP on the kinetics of serum protein synthesis in cultured mouse hepatoma cells

The mouse hepatoma cell line, Hepa, was cultured in the presence of either 1 mM N⁶, O^2′-dibutyryl cyclic AMP (Bt₂cAMP), 0.5 mM 3-isobutyl-1-methylxanthine, or 1 μg/ml cholera toxin. The synthesis and secretion of albumin, α-fetoprotein, and transferrin were elevated above controls by 24 h reaching two- to fourfold stimulations within 72 h. These effects were reversible and were specific for the serum proteins. The stimulation of serum protein synthesis was accompanied by a decrease in the rate of cell proliferation. Protein synthetic parameters were analyzed in Hepa cells 72 h after exposure to N⁶,O^2′-dibutyryl cyclic AMP. The cellular rate of albumin synthesis was increased fourfold and the relative rate of albumin synthesis was increased approximately threefold. N⁶,O^2′-Dibutyryl cyclic AMP did not affect the size distribution of either total Hepa polyribosomes or of albumin-synthesizing polyribosomes. The elongation rate on total mRNA and on albumin mRNA was decreased by approximately 40%. These results indicate that the rate of initiation of total Hepa mRNA and of albumin mRNA also decreased by 40%. The nonspecific nature of the N⁶,O^2′-dibutyryl cyclic AMP effect on Hepa protein synthetic parameters must be due to an alteration in the level of a common substrate, perhaps ATP. The specific threefold increase in the relative rate of albumin synthesis with no alteration in polyribosome sizes requires a threefold increase in the relative amount of functional albumin mRNA in Hepa cells. This prediction was confirmed by cell-free translation of Hepa polyribosomes.     

Studies of albumin binding to rat liver plasma membranes: Implications for the albumin receptor hypothesis

To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted ¹²⁵I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15 000 × g for 15 min), after repeated cycles of washing with buffer and re-centrifugation. ¹²⁵I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of ¹²⁵I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or ‘off-time’, as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase ¹²⁵I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor.