Changes in lipid pattern of HeLa cells exposed to immunoglobulin G and complement

1. Immunoglobulin G was isolated from sera of non-immunized rabbits or rabbits immunized with whole HeLa cell homogenate. The anti-HeLa immunoglobulin G and its Fab fragment precipitated the particulate 400000g-min. fraction of HeLa cell homogenate. 2. Immunoglobulin G from immunized or non-immunized rabbits and fresh or inactivated complement were added to HeLa cell cultures. Changes in the cell count and cellular contents of DNA, RNA, protein, total and individual phospholipids, cholesterol (and esters) and ganglioside were followed. 3. Addition of immunoglobulin G from non-immunized rabbits and guinea-pig serum (complement) caused a transient increase in DNA followed by a permanent increase in RNA, protein, dry weight and number of cells per culture. 4. Addition of anti-HeLa immunoglobulin G and active complement caused an increase in the cellular content of cholesterol, total phospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine greater than the increase of the controls and a decrease in the molar percentages of phosphatidylcholine and phosphatidylethanolamine as compared with the controls. 5. The cholesterol/phospholipid ratio remained constant. 6. The appearance of lysophosphoglycerides was transient, reaching a maximum 3hr. after addition of anti-HeLa immunoglobulin G. 7. The content of lysophosphoglycerides in HeLa cultures exposed to immunoglobulin G from non-immunized rabbits ranged from 50% to 30% of the values obtained from cultures exposed to the anti-HeLa immunoglobulin G and complement. 8. The changes in the lipid pattern of the HeLa cells were associated with the appearance of juxta-nuclear vacuoles in cells, but were apparently not specifically related to the presence of active complement.     

Incorporation of [32P]orthophosphate into brain-slice phospholipids and their precursors. Effects of electrical stimulation

1. The incorporation of [(32)P]phosphate into phospholipids was measured in slices cut from the pial surface of guinea-pig cerebral cortex; incorporation into the phosphorus of some water-soluble precursors of phospholipid was measured under similar conditions. 2. Slices subjected to overall electrical stimulation at a frequency of 5pulses/sec. differed from control slices in their pattern of phospholipid labelling. After 1hr. of stimulation, incorporation of [(32)P]phosphate into phosphatidylcholine, ethanolamine phospholipid and cardiolipin was respectively 54, 55 and 58% of the control value, and that into phosphatidylinositol was 186% of control. Phosphatidic acid labelling tended to increase with electrical stimulation, but the statistical significance of this change was marginal. Labelling of phosphatidylglycerol and di- and tri-phosphoinositides was not affected significantly by electrical stimulation. 3. Electrical stimulation of the tissue altered the specific radioactivities of water-soluble precursors of phospholipid. 4. The turnover rates of the phosphate groups of phospholipids were estimated approximately from the specific radioactivities of phospholipids and their precursors. Phosphatidylinositol (and its lipid-soluble precursors) showed the largest change in turnover rate in response to electrical stimulation of the tissue; the turnover rates of other lipids were also affected. Changes in the specific radioactivity of phospholipids did not correspond to changes in turnover in these experiments.     

Phospholipid exchange reactions within the liver cell

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.     

Phospholipid synthesis and exchange in isolated liver cells

1. The [(32)P]phosphate incorporated into the phospholipids of isolated rat hepatic cells is present in phosphatidic acid and to a smaller extent in phosphatidylinositol. 2. The ability to synthesize nitrogen-containing phospholipids is restored by adding a liver supernatant fraction, and it is suggested that the metabolic deficiency is caused by the leakage of cytoplasmic enzymes of the synthetase system from the cells. 3. Fortified cell preparations were pulse-labelled with [(32)P]phosphate, [Me-(14)C]choline, [2-(14)C]ethanolamine and [U-(14)C]inositol and the subsequent fate of the labelled microsomal and mitochondrial phospholipids followed. 4. A fall in the specific radioactivity of microsomal phospholipids and a rise in that of mitochondrial phospholipids is interpreted as providing evidence of a transfer of labelled phospholipid molecules from the synthetic site (endoplasmic reticulum) to the mitochondrial membranes in the intact cells. 5. The formation of the phospholipids of mitochondrial membranes is discussed.     

Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig.

Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.     

Compartmentation of phosphorylated precursors of phospholipid biosynthesis in cultured neuroblastoma cells

The continuous turnover of membrane phospholipids requires a steady supply of biosynthetic precursors. We evaluated the effects of decreasing extracellular Na+ concentration on phospholipid metabolism in cultured neuroblastoma (N1E 115) cells. Incubating cultures with 145 to 0 mM NaCl caused a concentration-dependent inhibition of [32P]phosphate uptake into the water-soluble intracellular pool and incorporation into phospholipid. Phospholipid classes were differentially affected; [32P]phosphate incorporated into phosphati-dylethanolamine (PE) and phosphatidylcholine (PC) was consistently less than into phosphatidylinositol (PI) and phosphatidylserine (PS). This could not be attributed to decreased phospholipid synthesis since under identical conditions, there was no effect on arachidonic acid or ethanolamine incorporation, and choline utilization for PC synthesis was increased. The effect of Na+ was highly specific since reducing phosphate uptake to a similar extent by incubating cultures in a phosphate-deficient medium containing Na+ did not alter the relative distribution of [32P]phosphate in phospholipid. Of several cations tested only Li+ could partially (50%) replace Na+. Incubation in the presence of ouabain or amiloride had no effect on [32P]phosphate incorporation into phospholipid. The differential effects of low Na+ on [32P]phosphate incorporation into PI relative to PC and PE suggests preferential compartmentation of [32P]phosphate into ATP in pools used for phosphatidic acid synthesis and relatively less in ATP pools used for synthesis of phosphocholine and phosphoethanolamine, precursors of PC and PE, respectively. This suggestion of heterogeneous and distinct pools of ATP for phospholipid biosynthesis, and of potential modulation by Na+ ion, has important implications for understanding intracellular regulation of metabolism.     

Phorbol ester-stimulated hydrolysis of phosphatidylcholine and phosphatidylethanolamine by phospholipase D in HeLa cells. Evidence that the basal turnover of phosphoglycerides does not involve phospholipase D

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulated the release of [3H]ethanolamine from HeLa cells prelabeled with [3H]ethanolamine within 2 min, and of [3H]choline from cells prelabeled with [3H]choline after a lag of 10-20 min. This result suggests that TPA activates phospholipase D. Propranolol alone or propranolol plus TPA stimulated phosphatidic acid (PA) labeling in cells prelabeled with [3H]hexadecanol. In the presence of ethanol, TPA stimulated the accumulation of labeled phosphatidylethanol (PEth); no PEth was formed in the absence of TPA. TPA-dependent PEth accumulation was not observed in cells pretreated with TPA to down-regulate protein kinase C, whereas propranolol-induced accumulation of PA was unaffected by TPA pretreatment. Incubation of prelabeled cells with propranolol alone caused a rapid loss of label and phospholipid mass from both phosphatidylethanolamine and phosphatidylcholine (PC) together with an accumulation of PA and phosphatidylinositol plus phosphatidylserine. When [3H]hexadecanol-prelabeled cells were pulse labeled with 32P to label nucleotide pools, propranolol induced the accumulation of both 3H- and 32P-labeled PA. When cells were prelabeled with lyso-PC double labeled with 3H and 32P, and incubated with propranolol, only 3H-labeled PA accumulated, indicating that the pathways involved in the basal turnover of PC resulted in the loss of 32P from the lipid. These results suggest that the basal turnover of phosphatidylethanolamine and PC involves the sequential actions of phospholipase C, diglyceride kinase, and PA phosphohydrolase.     

The catalytic center of glucose-6-phosphatase. HIS176 is the nucleophile forming the phosphohistidine-enzyme intermediate during catalysis

Glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis, is anchored to the endoplasmic reticulum by nine transmembrane helices. The amino acids comprising the catalytic center of G6Pase include Lys(76), Arg(83), His(119), Arg(170), and His(176). During catalysis, a His residue in G6Pase becomes phosphorylated generating an enzyme-phosphate intermediate. It was predicted that His(176) would be the amino acid that acts as a nucleophile forming a phosphohistidine-enzyme intermediate, and His(119) would be the amino acid that provides the proton needed to liberate the glucose moiety. However, the phosphate acceptor in G6Pase has eluded molecular characterization. To identify the His residue that covalently bound the phosphate moiety, we generated recombinant adenoviruses carrying G6Pase wild type and active site mutants. A 40-kDa [(32)P]phosphate-G6Pase intermediate was identified after incubating [(32)P]glucose 6-phosphate with microsomes expressing wild type but not with microsomes expressing either H119A or H176A mutant G6Pase. Human G6Pase contains five methionine residues at positions 1, 5, 121, 130, and 279. After cyanogen bromide cleavage, His(119) is predicted to be within a 116-amino acid peptide of 13.5 kDa with an isoelectric point of 5.3 (residues 6-121), and His(176) is predicted to be within a 149-amino acid peptide of 16.8 kDa with an isoelectric point of 9.3 (residues 131-279). We show that after digestion of a non-glycosylated [(32)P]phosphate-G6Pase intermediate by cyanogen bromide, the [(32)P]phosphate remains bound to a peptide of 17 kDa with an isoelectric point above 9, demonstrating that His(176) is the phosphate acceptor in G6Pase.     

Use of [125I]4'-iodoflavone as a tool to characterize ligand-dependent differences in Ah receptor behavior

We have synthesized [(125)I]4'-iodoflavone to study Ah receptor (AhR)-ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This radioligand allows the comparison of AhR-ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin. Specific binding of [(125)I]4'-iodoflavone with the AhR was detected as a single radioactive peak ( approximately 9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A approximately 6.7 S form of radioligand-bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [(125)I]4'-iodoflavone-AhR 6 S complex, compared with [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin, was recovered in nuclear extracts. A comparison of the ability of 4'-iodoflavone and TCDD to cause time-dependent translocation of AhR-yellow fluorescent protein revealed that 4'-iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [(125)I]4'-iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR.     

Probing into the function of the gene product responsible for glycogen storage disease type Ib

This study aimed at directly assessing glucose 6-phosphate (G6P) transport by intact rat liver microsomes. Tracer uptake from labeled G6P occurred with T(1/2) values that proved insensitive to unlabeled G6P or 100 microM vanadate, and could not be activated over background levels by intravesicular phosphate in the complete absence of G6P hydrolysis. [(32)P]Phosphate efflux was similarly unaffected by G6P or phosphate in the incubation medium. We conclude that the gene product responsible for glycogen storage disease type Ib is functionally distinct from the bacterial hexose phosphate transporter, which operates as an obligatory phosphate:phosphate or G6P:phosphate exchanger.     

Is high mobility group protein 17 phosphorylated in vivo? re-examination of the HeLa cell cycle data

When in vivo [32P] phosphate labeled HMG proteins from unsynchronized HeLa cells are separated by electrophoresis in acid-urea polyacrylamide gels, as opposed to separation in SDS-polyacrylamide, HMG 17 does not show any 32P incorporation. Likewise, no 32P radioactivity was found in HMG 17 protein isolated at different stages of the cell cycle from synchronized cells. By contrast, HMG 14 reveals a previously reported (Bhorjee, J.S. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6944-6948) cell cycle stage-specific dependent phosphorylation with maximum 32P radioactivity in the G2 phase relative to G1. Furthermore, HMG 14 is resolved into multiple electrophoretic forms as phosphoprotein in the acid-urea system. The results presented seriously question the data on the in vivo phosphorylation of HMG 17, and suggest that these be reevaluated.     

The turnover of myelin phospholipids in the adult and developing rat brain

1. Inorganic [(32)P]phosphate, [U-(14)C]glycerol and [2-(14)C]ethanolamine were injected into the lateral ventricles in the brains of adult rats, and the labelling of individual phospholipids was followed over 2-4 months in both a microsomal and a highly purified myelin fraction. 2. All the phospholipids in myelin became appreciably labelled, although initially the specific radioactivities of the microsomal phospholipids were somewhat higher. Eventually the specific radioactivities in microsomal and myelin phospholipids fell rapidly at a rate corresponding to the decline of radioactivity in the acid-soluble pools. 3. Equivalent experiments carried out in developing rats with [(32)P]phosphate administered at the start of myelination showed some persistence of phospholipid labelling in the myelin, but this could partly be attributed to the greater retention of (32)P in the acid-soluble phosphorus pool and recycling. 4. It is concluded that a substantial part of the phospholipid molecules in adult myelin membranes is readily exchangeable, although a small pool of slowly exchangeable material also exists. 5. A slow incorporation into or loss of labelled precursor from myelin phospholipids does not necessarily give a good indication of the rate of renewal of the molecules in the membrane. As presumably such labelled molecules originate by exchange with those in another membrane site (not necessarily where synthesis occurs) it is only possible to calculate the turnover rate in the myelin membrane if the behaviour of the specific radioactivity with time of the phospholipid molecules in the immediate precursor pool is known.     

Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide-binding proteins from HeLa S3 cells

Nucleoside-diphosphate (NDP) kinase-associated [alpha-32P]GTP-incorporating proteins from HeLa S3 cells have been biochemically characterized. Two distinct NDP-kinases (F-I and F-II) had been partially purified from HeLa S3 cells by Sephacryl S-300 gel filtration and DEAE-cellulose column chromatography. The [alpha-32P]GTP-incorporating proteins (approx. Mr 20,000) could be separated from NDP-kinases (approx. Mr 80,000) by 5-25% glycerol density-gradient centrifugation analysis after treatment with 7 M urea in the presence of 1 mM EDTA. [alpha-32P]GTP incorporation into these two proteins (G1 and G2) from NDP-kinases required 5 mM Mg2+ and was highly inhibited by either GDP or GTP analogues, such as guanylyl imidodiphosphate and guanylyl methylenediphosphate. [3H]GDP, but no other nucleoside 5'-diphosphates, was also bound to these two proteins in the presence of Mg2+ (5 mM). Moreover, incubation of [alpha-32P]GTP with either G1 or G2 in the presence of Mg2+ (5 mM) resulted in the formation of [32P]GDP and Pi. The data presented here indicated that the guanine nucleotide-binding activity, the GTPase activity, and the molecular weight (approx. Mr 20,000) of NDP-kinase-associated proteins from HeLa S3 cells are similar to those reported for ras oncogene products (p21 proteins).     

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled iodoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3′,5′-(cyclic)-adenosine monophosphate

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.     

Phosphorylation of high-molecular-weight membrane protein species in Chinese-hamster ovary cells in culture: effect of 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate plus testosterone (Short Communication)

Growth of Chinese-hamster ovary cells in [(32)P]phosphate and [(3)H]leucine and subsequent assay of the plasma membranes reveals phosphorylation in two protein regions corresponding to molecular weights of 280000 and 195000. Culture in the presence of the 6-N,2-O'-dibutyryl derivative of cyclic AMP plus testosterone does not stimulate [(32)P]-phosphate incorporation, but determines a modification in the qualitative pattern of phosphorylation.     

A complement-resistant HeLa cell line (T638) is blocked at the step of C3 deposition

A complement-resistant line of HeLa cells (T638) was derived by serial passage of complement-susceptible HeLa cells in anti-beta 2-microglobulin (b2m) antiserum and complement. The T638 line maintained stable complement resistance when passed for an additional 1500 generations in the absence of antiserum and complement. T638 cells expressed equivalent levels of cell-associated b2m as did the parent HeLa cell line. Furthermore, T638 cells were resistant to killing by complement and anti-HeLa antiserum with specificity for molecules other than b2m. These results indicate that the resistance of T638 cells does not simply reflect loss of anti-b2m binding antigens. We next investigated the mechanism of resistance of T638 cells to complement-mediated killing. Antibody-sensitized HeLa and T638 cells both consumed CH50 activity completely from normal human serum; cytotoxicity was not mediated via the alternative complement pathway. HeLa and T638 cells caused equivalent utilization of C4 from normal human serum in the presence of antibody. Consumption of C2, greater with T638 than with HeLa cells during incubation in serum, was complete when cells bearing purified C1 and limited C4 were incubated with C2. T638 cells bound more 3H-C4 than HeLa cells during incubation in serum, but binding of 3H-C3 by T638 cells was fourfold to fivefold less than by HeLa cells. Finally, we investigated the rate of decay in the capacity of C142 on HeLa and T638 to cleave and deposit 3H-C3. The T1/2 for decay of C142-mediated binding of 3H-C3 on HeLa was 3.9 min, whereas minimal C3 deposition was detected on T638 cells at all time points. These results show that T638 cells evade complement-mediated lysis despite activating early components of the classical complement pathway. The mechanism of resistance is a failure to form an effective C3 convertase.     

Turnover of mitochondrial components of normal and essential fatty acid-deficient rats

1. Essential fatty acid (EFA)-deficient and control rats were injected intraperitoneally with [(32)P]phosphate, l-[(35)S]methionine and [2-(14)C]acetate. The animals were killed at various time-intervals after injection and their liver mitochondria fractionated into soluble protein, insoluble protein, and lipid. 2. The (35)S was assayed in the protein fractions and (32)P and (14)C were assayed in the lipid fraction. Curves of log (specific activity) plotted against time were prepared for the different fractions. 3. There was no significant difference between the insoluble protein results for control and EFA-deficient animals, both sets of results indicating the presence of a single component of half-life 9 days. 4. There was no significant difference between the soluble protein results for the two sets of animals and both sets of results indicated the presence of at least two components. 5. The [(32)P]-phospholipid results indicate that in the control animals the liver mitochondrial phospholipids contain components of half-life 1.6 and 10 days whereas the mitochondrial phospholipids of the EFA-deficient animals contain components of half-life 3 and 29 days. 6. The specific activity of mitochondrial [(14)C]phospholipid initially fell rapidly in both groups of animals, but after 17 days there was no further significant decrease. A fast component with maximum half-life 2-4 days was clearly demonstrated for both groups of animals. Whether or not these results also indicate the presence of a very long-lived mitochondrial phospholipid is discussed.     

Intracellular generation of sphingosine 1-phosphate in human lung endothelial cells: role of lipid phosphate phosphatase-1 and sphingosine kinase 1

Sphingosine 1-phosphate (S1P) regulates diverse cellular functions through extracellular ligation to S1P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S1P to generate intracellular S1P. We, therefore, examined the role of lipid phosphate phosphatase (LPP)-1 and sphingosine kinase1 (SphK1) in converting exogenous S1P to intracellular S1P. Exposure of (32)P-labeled HPAECs to S1P or sphingosine (Sph) increased the intracellular accumulation of [(32)P]S1P in a dose- and time-dependent manner. The S1P formed in the cells was not released into the medium. The exogenously added S1P did not stimulate the sphingomyelinase pathway; however, added [(3)H]S1P was hydrolyzed to [(3)H]Sph in HPAECs, and this was blocked by XY-14, an inhibitor of LPPs. HPAECs expressed LPP1-3, and overexpression of LPP-1 enhanced the hydrolysis of exogenous [(3)H]S1P to [(3)H]Sph and increased intracellular S1P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-1 by siRNA decreased intracellular S1P production from extracellular S1P but had no effect on the phosphorylation of Sph to S1P. Knockdown of SphK1, but not SphK2, by siRNA attenuated the intracellular generation of S1P. Overexpression of wild type SphK1, but not SphK2 wild type, increased the accumulation of intracellular S1P after exposure to extracellular S1P. These studies provide the first direct evidence for a novel pathway of intracellular S1P generation. This involves the conversion of extracellular S1P to Sph by LPP-1, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S1P by SphK1.     

Manifold effects of sodium butyrate on nuclear function. Selective and reversible inhibition of phosphorylation of histones H1 and H2A and impaired methylation of lysine and arginine residues in nuclear protein fractions

In addition to its known effect in suppressing the deacetylation of the nucleosomal core histones, sodium butyrate in the concentration range 0.5 to 15 mM causes a selective inhibition of [32P]phosphate incorporation into histones H1 and H2A of cultured HeLa S3 cells. No commensurate general inhibition of phosphorylation is seen in the non-histone nuclear proteins of butyrate-treated cells, but phosphorylation patterns are altered and 32P-uptake may be stimulated, as well as inhibited, depending upon the protein fraction analyzed. The degree of inhibition of histone phosphorylation in intact cells increases progressively as the butyrate concentration is raised from 0.5 to 15 mM. The effect is time-dependent and fully reversible. Butyrate has no effect on the kinetics of phosphate release from previously phosphorylated histones of cultured cells, nor does it significantly alter the rate of dephosphorylation of 32P-labeled histone H1 by endogenous phosphatases in vitro. Despite the suppression of [32P]phosphate incorporation into histones H1 and H2A of butyrate-treated cells, Na-butyrate does not inhibit the in vitro activities of either type I or type II cyclic AMP-dependent protein kinases, or the cAMP-independent H1 kinase associated with cell cycle progression. This suggests that the butyrate effect on histone phosphorylation in vivo is indirect and may involve an alteration in substrate accessibility or a modulation of systems affecting kinase activities. The poly(ADP)-ribosylation of HeLa histones is not inhibited by 5 mM Na-butyrate. Cells exposed to butyrate show an impaired methylation of lysine and arginine residues in their histones and nuclear hnRNP particles, respectively.     

The biochemistry of virus-induced cell fusion. Changes in membrane integrity

1. Phospholipids prelabelled with [(14)C]acetate, [(32)P]phosphate, [(3)H]- or [(14)C]-choline or [(3)H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [(3)H]fucose, [(14)C]galactose or [(3)H]glucosamine. Less than 1nmol of lysophosphatidylcholine/10(7) cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K(+) leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca(2+) into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G(1) and S periods of the cell cycle. 5. Leakage is inhibited by Ca(2+), but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.