Rapid transport of fatty acids from rat liver endothelial to parenchymal cells after uptake of cholesteryl ester-labeled acetylated LDL

Acetylated low-density lipoprotein (acetyl-LDL) radiolabeled in the oleate moiety of cholesteryloleate was injected into rats. Isolation of the various liver cell types at different times after acetyl-LDL injection by a low-temperature procedure allowed the intrahepatic metabolism of the oleate moiety to be followed in vivo. The cholesteryloleate radioactivity is rapidly cleared from the circulation and at 5 min after injection recovered into parenchymal and endothelial liver cells, mainly as cholesteryloleate ester. At longer time intervals after injection, the amount of cholesteryl esters associated with the endothelial cells was sharply decreased and the [14C]oleate was redistributed within the liver and mainly recovered in the parenchymal cells. The cholesteryl ester initially directly taken up by the parenchymal cells was also rapidly hydrolysed but, in contrast to the endothelial cells, the [14C]oleate remained inside the cells and was incorporated into triacylglycerols and phospholipids. The 14C radioactivity in parenchymal cells taken up between 5 and 30 min after injection of the cholesteryl [14C]oleate-labeled acetyl-LDL (transported as oleate from endothelial cells), followed a similar metabolic route as the amount which was directly associated to parenchymal cells. The data indicate that the liver and, in particular, the liver endothelial cell has the full capacity to rapidly catabolize modified lipoproteins. In this catabolism, the liver functions as an integrated organ in which fatty acids, formed from cholesteryl esters in endothelial cells, are rapidly transported to parenchymal cells, indicating the concept of metabolic cooperation between the various liver cell types.     

Properties of the complexes of riboflavin 3',5'-bisphosphate and the apoflavodoxins from Megasphaera elsdenii and Desulfovibrio vulgaris

Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3',5'-bisphosphate. Several biochemical and biophysical properties of the complexes have been investigated and the results are compared with the properties of the native proteins. The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for the native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone-hydroquinone transition is about 20 mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein-bound artificial prosthetic group and the protein-bound natural prosthetic group are very similar. In both the oxidized and in the fully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15N NMR. 31P-NMR studies show that the 5'-phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3'-phosphate group in riboflavin 3',5'-bisphosphate is monoanionic or even neutral when bound to the protein. The 3'-phosphate group is also close to or on the surface of the protein. Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3',5'-bisphosphate which is 10 times higher as compared to Megasphaera elsdenii apoflavodoxin (Ka = 10(8) M-1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3'5'-bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3',5'-bisphosphate analog gives a value (Kd approximately equal to 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649-655]. The results are discussed in the light of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins and flavoproteins, pp. 493-496, Walter de Gruyter, Berlin].     

Anti-idiotypic treatment of BALB/c mice induces CRIa-bearing suppressor cells with altered Igh-restricted function

The ABA-specific antibody response of A/J mice (Igh Ie) is dominated by the CRIa idiotype. In contrast, BALB/c mice (Igh Ia) do not produce CRIa-bearing anti-ABA antibodies after antigenic challenge. We have shown previously that treatment with rabbit anti-CRIa (R-anti-CRIa) induces the expression of "CRIa-like" anti-arsonate antibodies in BALB/c mice. In the present report, we demonstrate that R-anti-CRIa treatment enables BALB/c mice to respond to A/J ABA-specific first-order suppressor molecules (TsF1). Manipulated BALB/c also produced CRIa bearing ABA-specific immune response. Thus, R-anti-CRIa treatment induces a change in the characteristic Igh restriction pattern typically seen in this system. These data suggest that Igh restriction in the ABA-specific T suppressor cell pathway is the result of CRIa+ dominance in the T suppressor cell response of A/J mice. The effectiveness of idiotypic manipulation in inducing the expression of a given idiotype at both the B cell and T suppressor cell levels is discussed.     

Reactivity and fate of benzene and formaldehyde in culture medium with and without fetal calf serum; relevance to in vitro mutagenicity testing

Gas chromatographic-mass spectrometric analyses were performed to determine the reactivity and fate of benzene (BEN) and formaldehyde (FA) in culture medium. BEN (solubility in water: 500 ppm) does not react with culture medium, either with or without fetal calf serum, but its volatility, even in closed vials, is so great that 90% of a 250-ppm solution is lost to the head space after 1 h at 24°C. FA, as a 37% aqueous solution, is a complex mixture that changes composition after 15-min incubation at 38°C. FA is extremely reactive in culture medium containing fetal calf serum, and is much less reactive with medium components in the absence of serum. There is a dramatic increase in the number of daughter products in FA-treated medium over time, such that those seen immediately after FA is added to medium have been replaced after 60-min incubation (38°C in closed vials) by many other interaction products. Methods ensuring maximum solubilization and minimal volatilization of BEN during exposure are essential for obtaining reproducible data on the mutagenic potential of BEN. The volatilization of FA from stock formalin solutions, and, more importantly, the interaction product(s) formed by this highly reactive compound with medium components, especially those in serum, are probably the critical aspects of an effective testing protocol for FA.     

Visualization of the interaction of native and modified low density lipoproteins with isolated rat liver cells

Morphological characteristics of the interaction of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) with rat liver cells are described. These liver cell types are mainly responsible for the catabolism of these lipoproteins in vivo. Isolated rat liver Kupffer, endothelial, and parenchymal cells were incubated with LDL or AcLDL conjugated to 20 nm colloidal gold. LDL was mainly internalized by Kupffer cells, whereas AcLDL was predominantly found in endothelial cells. Kupffer and endothelial cells displayed different morphological characteristics in the processing of these lipoproteins. Kupffer cells bound LDL at uncoated regions of the plasma membrane often at the base of pseudopodia, and internalized the particles via small smooth vesicles. These uptake characteristics differ from the classical LDL uptake pathway, as described for other cell types, and may be related to the unique recognition properties of the receptor of Kupffer cells as observed in biochemical studies. Liver endothelial cells bound AcLDL in coated pits, followed by rapid uptake. Uptake proceeded through small coated vesicles, and after 5 min of incubation large (600-1200 nm) electron-lucent vacuoles (endosomes) with AcLDL-gold particles arranged along the membrane region were present. The endosomes were often associated closely with the cell membrane which might enable direct recycling of AcLDL receptors. These observations might explain the high efficiency of these cells in the processing of modified LDL in vivo.     

Lysis of autologous tumor cells by blood lymphocytes tested at the time of surgery

Summary Lymphocyte-mediated lysis of autologous tumor cells (autologous lymphocyte cytotoxocity ALC) was tested at the time of surgery in 108 patients (46 squamous cell carcinomas of the lung, 25 adenocarcinomas of the lung, 19 soft tissue sarcomas and 18 osteosarcomas). The clinical course of these patients in relation to the test results has been published previously. The group was evaluated again after an extended observation time, now with a mean of 80.2 months (range 36–108). The test was rarely positive in patients with metastasis (2 out of 28 experiments).There was a correlation between the ALC results and the postsurgical clinical course for patients without detectable metastasis in that (1) a negative test was invariably a bad prognostic sign, i.e., all 32 patients with negative ALC died within 3 years (mean survival time 16.1 months). (2) The remission and survival times were longer for the ALC positive patients (p<0.001). (3) All 37 individuals who are alive at present without recurrence belong to the reactive group.The ALC results correlated with the clinical course in 88% of patients. The correlation was highest for the groups of soft tissue sarcoma and adenocarcinoma of the lung. There was no correlation between killing of K562 cells and ALC, or between lymphoproliferative response to PHA and ALC reactivity.     

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.     

Effect of apolipoproteins E and C-III on the interaction of chylomicrons with parenchymal and non-parenchymal cells from rat liver.

[3H]Triacylglycerol-labelled chylomicrons were isolated from intestinal lymph, obtained from rats made hypolipidaemic by treatment with pharmacological amounts of 17 alpha-ethynyloestradiol. Oestrogen treatment results in a large reduction in the content of apolipoproteins (apo) E and C of lymph chylomicrons. Upon incubation in vitro with freshly isolated parenchymal and non-parenchymal cells the apo E-, apo C-poor chylomicrons became readily cell-associated. With increasing chylomicron concentrations this cell-association was saturable and half-maximal cell-association was achieved at about 0.55 mg of triacylglycerol/ml. The cell-association was time- and temperature-dependent. A more than 90% inhibition of the cell-association of the [3H]triacylglycerol moiety was observed with both parenchymal and non-parenchymal cells when pure apo C-III (12.6 micrograms/mg of triacylglycerol) was incorporated into the chylomicrons. These data indicate that apo E-, apo C-poor chylomicrons are bound to both parenchymal and non-parenchymal liver cells at a high-affinity site of limited capacity and that binding to this site is strongly inhibited by apo C-III. With apo C-III-enriched chylomicrons simultaneous determination of the cell-association of the 125I-apo C-III and the [3H]triacylglycerol moiety indicated that more 125I-apo C-III becomes associated to the cells than expected on the basis of [3H]triacylglycerol radioactivity measurements. It is suggested that upon cell-association of apo C-III its binding to the chylomicron particles is lost. Consequently the occupation of the cellular recognition site by apo C-III prevents further chylomicron binding and thus leads to a decrease of the cell-association level of the [3H]triacylglycerol moiety. Apo E enrichment of the chylomicrons led to an increased cell-association rate with parenchymal cells and to a marked increase of the cell-association level with non-parenchymal cells. The cell-association of the apo E radioactivity followed closely the [3H]triacylglycerol radioactivity, indicating that the particle-apo E complex is bound as a unity. The apo E effects were opposed by apo C-III. With apo E-, apo C-III-enriched chylomicrons more 125I-apo E became associated with the cells than could be expected on the basis of the [3H]triacylglycerol measurements. It is concluded that apo C-III can weaken the interaction of apo E with the chylomicrons leading to the cell-association of free apo E. It appears that subtle changes in the apo E and/or apo C-III content of chylomicrons can influence the interaction with both parenchymal and non-parenchymal liver cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

NUTRITION OF PANDORINA MORUM1,2

A defined medium was developed for 3 strains of Pandorina morum. The strains tested required no vitamins or other organic compounds. The optimal initial pH was between 7.0 and 8.0. Various carbon sources were tested, and only glycolate and acetate appreciably stimulated growth. Mixotrophic growth in the light was stimulated by glycolate in all 3 strains, and by acetate in strains 880 and N76-6. Only strain N76-6 utilized acetate for heterotrophic growth in the dark. Thirty strains of P. morum of world-wide distribution were surveyed for mixotrophic and heterotrophic growth with acetate. All were found to fit 1 of 3 classes with respect to acetate metabolism: (1) no effect in light or dark; (2) stimulation of growth in light only; (3) stimulation of growth in light and dark.     

Isolation and analysis of (Gp)nXp sequences of rat liver 5S RNA by means of restricted ribonuclease T2 hydrolysis

Essentual difficulties arise when base number in oligoguanylic blocks and location of these blocks along the polynucleotide chain need to be determined in the course of determination of the nucleotide sequences in ribonucleic acids. To overcome this difficulty it is suggested to take advantage of a recently discovered resistance of phosphodiester bond between kethoxalated G and its 3′-neighbour against T₂ RNase hydrolysis 1,2. The approach is illustrated by analysis of 5S RNA from rat liver. Sequences of general formula (Gp)_nXp were isolated from T₂ RNase hydrolysate of 5 S RNA rapidly and quantitatively. The information obtained greatly facilitates the whole procedure of sequencing. It is expected that the method proposed would be effective for analysis of 5 S and 4 S RNA and for highmolecular weight fragments of ribosomal and viral RNAs.