Liver Cell Homogenates Research Articles

Electrochemical simulation of cocaine metabolism-a step toward predictive toxicology for drugs of abuse.

Knowledge of the metabolic pathways and biotransformation of the most popular drugs, such as cocaine, amphetamine, morphine and others, is crucial for the elucidation of their possible toxicity and mechanism of action in the human body. In vitro studies on metabolism are mainly based on the incubation of drugs with liver celL homogenate and utilizing Living animals. These methods need to be followed by isolation and detection of metabolic products, which makes these techniques time-consuming and technically demanding. We show here that the oxidative metabolism that occurs in the liver cells and is mainly caused by cytochrome P450 can be successfully mimicked with the electrochemical system [EC] connected on-line with electrospray ionization mass spectrometry. Cocaine was chosen as a model drug for these studies and was analyzed with a previously described system under various conditions using the boron-doped diamond working electrode. The results were compared with the number of metabolites generated by a standard procedure based on the reaction with the rat Liver microsomes. Two electrochemical products of cocaine oxidation were created, of which one was a natural metabolite of cocaine in the human body-norcocaine. The EC provides a promising platform for the screening of the addictive drug phase I metabolism. The metabolites can be directly analyzed by mass spectrometry or collected and separated by Liquid chromatog- raphy. No Liver cell homogenate or microsome is necessary to generate these metabolites, which simplifies separation of the mixtures and reduces time and costs of all experiments.

Aminoguanidine partially prevents the reduction in liver pyruvate kinase activity in diabetic rats.

Background:Low molecular weight aldehydes and carbonyl compounds which are derived from glucose metabolism are prevalent in diabetic plasma. These compounds react to amino groups of Lys and Arg and lead to the formation of advanced glycation end products (AGEs). This modification changes the function of the proteins. The present study aimed to survey the effect of diabetes on rat liver pyruvate kinase activity and to show the inhibitory effect of aminoguanidine (AG).Materials and Methods:Male Wistar rats (n = 18, 6 to 8 weeks old) were divided randomly in three groups: the first group as control; second and third groups were induced diabetes using streptozocin. Third group received AG orally for 8 weeks after diabetes induction. Liver cell homogenate was prepared from all studied groups and L-type pyruvate kinase was separated from the homogenate. Pyruvate kinase activity was determined in both liver cell homogenate and extracted L-type PK. The PK activity was compared in all samples between groups.Results:PK activity in isolated form and in liver cell homogenate was lower in diabetic rats as compared to control group. AG-treated group showed higher PK activity compared to untreated diabetic group; however, the difference was not significant. Non-significant difference in PK activity between AG-treated diabetic and non-diabetic (control) group indicated the inhibitory effect of AG in glycation of PK.Conclusion:The obtained results showed PK activity decreased in diabetic rats and AG can partially prevent the reduction in PK activity.

D-glucose- and 3-O-methyl-D-glucose-induced upregulation of selected genes in rat hepatocytes and INS1E cells: Re-evaluation of the possible role of hexose phosphorylation

The biochemical events involved in the upregulation of selected glucose‑responsive genes by 3‑O‑methyl‑D‑glucose (3‑MG) remain to be elucidated. The present study mainly aimed to re‑evaluate the possible role of 3‑MG phosphorylation in the upregulation of the thioredoxin interacting protein (TXNIP) and liver pyruvate kinase (LPK) genes in rat hepatocytes and INS1E cells. TXNIP and LPK transcription was assessed in rat liver and INS1E cells exposed to a rise in D‑glucose concentration, 2‑deoxy‑D‑glucose (2‑DG), 3‑MG and, when required, D‑mannoheptulose. The phosphorylation of D‑[U‑14C]glucose and 3‑O‑[14C]methyl‑D‑glucose (14C-labeled 3-MG) was measured in rat liver, INS1E cell and rat pancreatic islet homogenates. The utilization of D‑[5‑3H]glucose by intact INS1E cells was also measured. In rat hepatocytes, a rise in the D‑glucose concentration increased the TXNIP/hypoxanthine‑guanine phosphoribosyl transferase (HPRT) and LPK/HPRT ratios, while 2‑DG and 3‑MG also increased the TXNIP/HPRT ratio, but not the LPK/HPRT ratio. In INS1E cells, the TXNIP/HPRT and LPK/HPRT ratios were increased in response to the addition of D‑glucose, 2‑DG and 3‑MG. Furthermore, D‑mannoheptulose abolished the response to D‑glucose and 2‑DG, but not to 3‑MG, in these cells. Liver cell homogenates catalyzed the phosphorylation of 3‑MG to a modest extent, whilst INS1E and rat pancreatic islet cell homogenates did not. Moreover, 3‑MG marginally decreased D‑glucose phosphorylation in INS1E cell homogenates but not in liver cell homogenates. D‑[5‑3H]glucose utilization by intact INS1E cells was decreased by 2‑DG, but not by 3‑MG. These findings reinforce the view that the upregulation of the TXNIP and LPK genes induced by 3‑MG is not attributable to its phosphorylation or any favorable effect on D‑glucose metabolism.

Proteomic Analysis of Ionizing Radiation-Induced Proteins at the Subcellular Level

Irradiation induces a series of liver diseases. However the molecular mechanisms involving in the process of liver diseases induced by irradiation are still unclear. Subcellular proteomics provides a method to understand regional differences in protein expression levels. With accumulating evidence in the literature that new proteins are implicated in radiation response, in the present study, C57BL/6 mice were treated with irradiation, liver cell homogenates were subfractionated by differential ultracentrifugation into nuclei, mitochondria and cytosol, which were subjected to 2-DE to generate the proteomic maps of these fractions. The differentially expressed proteins in the nuclei, mitochondria and cytosol compartment of liver at 24 and 48 h after exposure to 20 Gy irradiation compared to control were identified by MALDI-TOF MS respectively. Total 37 proteins at 24 h and 29 proteins at 48 h were matched with known proteins after database searching in nuclei, mitochondria and cytosol, respectively, among which nine proteins exhibited changes at both time points. Most of these proteins are involved in antioxidant response, energy metabolism, molecular chaperones and inflammatory response. More antioxidant-associated proteins were induced at 48 h than 24 h. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting further validated 2-DE results of two of these proteins. It is feasible that the differential proteins identified in this study have a biological significance and may provided clues for understanding the mechanism of injury in liver induced by irradiation.

Synthesis and biological activation of an ethylene glycol-linked amino acid conjugate of cyclic cidofovir

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs ( 3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P–O ester bond were synthesized and their pH-dependent stability ( 3 and 4), potential for in vivo reconversion to drug ( 3), and oral bioavailability ( 3) were evaluated. The prodrugs were stable in buffer between pH 3 and 5, but underwent rapid hydrolysis in liver ( t 1/2 = 3.7 min), intestinal ( t 1/2 = 12.5 min), and Caco-2 cell homogenates ( t 1/2 = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4× more active than ganciclovir (IC 50 value, 0.68 μM vs 3.0 μM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed.

Proteomic profiles of induced hepatotoxicity at the subcellular level

In the present study proteomes of liver samples were analyzed after administration of phenobarbital (PB) or 3-methylcholantrene (3-MC) to mice. Liver cell homogenates were subfractionated by differential ultracentrifugation into cytosol and microsomes, which were subjected to 2-DE to generate the proteomic maps of these fractions. 2-DE yielded 1100 and 800 protein spots for microsomes and cytosol, respectively. General trends of the fraction-specific alterations after 3-MC or PB treatment were evaluated using the Student's t-test and the principal component analysis (PCA). According to the PCA-derived data, the microsomal changes after 3-MC and PB treatment were quite similar. However, in the case of the cytosol data, the specificities of 3-MC- and PB-induced responses could be clearly distinguished from each other. Protein spots, whose expression levels differed from control, were identified by MALDI-TOF PMF. Proteomic studies such as those reported herein can be useful in identifying the molecular-based toxicity of lead drug candidates.

Fructokinase activity in rat liver, ileum, parotid gland, pancreas, pancreatic islet, B and non-B islet cell homogenates

The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously documented. However, no information was so far available on the activity of this enzyme in islets relative to that in other tissues and on the respective contribution of insulin-producing B cells and non-B islet cells. The present study provides such an information. The activity of fructokinase, as assessed by the phosphorylation of 1.0 mM D-fructose, was compared to that of hexokinase isoenzyme(s), as measured in the presence of 1.0 mM D-glucose, and further characterized by its heat-resistance, K+ dependency and resistance to the inhibitory action of D-mannoheptulose. As judged from the results obtained in heated homogenates, the activity of fructokinase, expressed relative to protein content (nmol/min per mg protein) was highest in liver (21.5 +/- 2.5; n = 11) and lowest in parotid gland (0.16 +/- 0.09; n = 3), with in-between values in ileum (2.45 +/- 0.53; n = 3), pancreas (0.82 +/- 0.11; n = 11) and pancreatic islets (0.46 +/- 0.07; n = 6). The paired ratio between fructokinase and hexokinase isoenzyme activity was also highest in liver (548 +/- 45%; n = 8) and lowest in parotid gland (0.93 +/- 0.52%; n = 3). Such a ratio was not significantly different in pancreas, islets and purified B or non-B islet cells, with an overall mean value of 2.57 +/- 0.46% (n = 12). The present findings thus unambiguously document the presence of fructokinase activity in all cell types under consideration, except possibly parotid cells, with the following hierarchy: liver > ileum > pancreas. Relative to paired hexokinase activity, no obvious difference was found for fructokinase activity in B versus non-B islet cells.

Synthesis andin vitroEnzyme Hydrolysis of Trioxadiaza- and Tetraoxadiaza-Crown Ether-Based Complexing Agents with Disposable Ester Pendant Arms

New disposable ester derivatives of the complexing agents N,N′-bis(carboxymethyl)piperazine, -homopiperazine, -1,7-diaza-15-crown-5 and -1,10-diaza-18-crown-6 were synthesized with a variety of synthetic methods and fully characterized. Hydrolytic properties of the pendant arms were studied under different pH conditions as well as in the presence and absence of porcine liver esterase enzyme and approximate hydrolysis half lives were determined by 1H NMR technique. In vitro studies on pig liver cell homogenates and living thin chicken liver slices proved that the selected double ester 4b can penetrate liver tissues spontaneously, liberate the free complexing agent N,N′-bis(carboxymethyl)-18-ane-N2O4 (ODDA) inside the cells and are potentially capable of removing lead and other toxic metal ions from the liver.

Hemin-mediated restoration of allylisopropylacetamide-inactivated CYP2B1: a role for glutathione and GRP94 in the heme-protein assembly.

Administration of the cytochrome P450 (P450) suicide inactivator allylisopropylacetamide (AIA) to phenobarbital (PB)-pretreated rats results in rapid and marked inactivation of several liver endoplasmic reticulum (ER)-bound P450s. A few of these such as CYP2B1, inactivated due to AIA-mediated prosthetic heme N-alkylation, can be structurally and functionally restored nearly completely by exogenous hemin in vivo or in vitro. Such in vitro hemin-mediated reassembly is unsuccessful with purified AIA-inactivated CYP2B1 and, as shown herein, is not very effective even when heme is incubated with just the corresponding liver microsomes that contain the reconstitutable CYP2B1 protein, thereby implicating a requirement for additional factors provided by the intact liver cell homogenates, ER, and/or cytosol. Using various approaches that include high-performance liquid chromatographic fractionation of the liver cytosolic subfraction as well as chemical and immunological probes such as the Hsp90/GRP94-specific inhibitor geldanamycin (GA) and polyclonal anti-GRP94 antibodies, respectively, we now demonstrate that the in vitro hemin-mediated reassembly of heme-stripped microsomal CYP2B1 requires GSH as well as the ER chaperone GRP94, but not the cytosolic chaperone heat shock protein 90. It remains to be determined whether GSH acts directly or indirectly, via a putative ER thiol reductase, to maintain the conserved active site cysteine-thiol (Cys436 in CYP2B1) in a reduced state, competent for heme binding and repair.

Isolation and characterization of metabolites of centpropazine in rat liver, intestine, and red blood cell homogenates

The potential sites for metabolism of centpropazine (CPZ) (an antidepressant) were evaluated in male Sprague‐Dawley rats. The isolation and identification of the major metabolites formed in the presence of rat liver S9 fraction, intestine, and red blood cells under aerobic conditions were performed using high‐performance liquid chromatography and electrospray ionization mass spectrometry. CPZ was found to be extensively metabolized to seven possible metabolites by liver S9 fraction in the presence of a nicotinamide adenine dinucleotide phosphate generating system at 37°C. Both intestinal wall and red blood cells were also found to metabolize the compound. This metabolite structure was confirmed by comparison with that of its synthetic standard. The drug was stable in intestinal contents. On the basis of our finding, we propose the in vitro metabolic pathways for CPZ. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2067–2075, 2002

Induction of zebrafish (Brachydanio rerio) P450 in vivo and in cell culture.

1. Induction of zebrafish P450 by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was studied in liver tissue, primary liver cell culture and multipassage cell culture derived from zebrafish haploid and diploid embryos and liver. 2. TCDD induced two hepatic proteins (54 and 50 kDa) in vivo which were recognized by anti-trout P4501A1 IgG. The 54-kDa protein was induced by TCDD in primary and multipassage hepatocyte cultures and in haploid and diploid embryo-derived cells. The proteins in liver homogenates were not induced by aqueous exposure of zebrafish to beta-naphthoflavone (BNF). 3. Homogenates of zebrafish liver, cultured hepatocytes and embryo-derived cells also exhibited increased ethoxyresorufin O-deethylase (EROD) and 7-12-dimethyl-benz[a]anthracene (DMBA) hydroxylase activity following TCDD exposure.

Radiation Hepatology of the Rat: Parenchymal and Nonparenchymal Cell Injury

Cellular changes that occur within the liver before and during liver fibrosis have been studied. At various times (0-90 days) after gamma irradiation (0-25 Gy) of the whole liver, hepatocytes and liver nonparenchymal cells were isolated by enzymatic dissociation of the liver and differential centrifugation. Differential cell counts were done to quantify the yield of hepatocytes and nonparenchymal cells. Hepatocyte function in vitro was assayed by the uptake of rose bengal and compared with the clearance of the dye in vivo. Intracellular alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were used to estimate the effect of radiation on the phenotypic expression of hepatocytes. Gene expression of transforming growth factor beta 1 (TGF-beta 1), a cytokine which has been found to play a role in hepatic fibrosis, was measured by 32P-cDNA:mRNA hybridization of poly(A+) mRNA isolated from purified populations of hepatocytes and nonparenchymal cells. Morphologically, hemorrhages were evident in perfused livers 28 days after 25 Gy whole-liver irradiation. This injury was preceded by a significant increase in liver nonparenchymal cells at 2 weeks, the earliest time after irradiation at which measurements were made. Significant increases in liver nonparenchymal cells occurred at doses greater than 10 Gy and persisted throughout the follow-up period after irradiation. The lowest dose studied (5 Gy) inhibited both normal growth of the liver and the associated increase in the number of hepatocytes. Doses greater than 10 Gy resulted in a dose-dependent decline in liver mass and hepatocytes. Viable hepatocytes isolated from irradiated dysfunctional livers, with respect to clearance of rose bengal, were functionally normal in vitro. A dose-dependent decline in the ALT and AST contents of liver cell suspensions and homogenates was observed but the relative decline in ALT was greater, resulting in lower ALT/AST ratios. This decline in the ALT/AST ratios occurred at doses (< 15 Gy) which caused no loss of hepatocytes, suggesting possible conversion of ALT-rich periportal hepatocytes to ALT-poor hepatocytes due to changes in the microenvironment and/or aging of the cell population. TGF-beta 1 mRNA was detected mainly in nonparenchymal cells, and radiation preferentially enhanced the TGF-beta 1 gene expression in these cells. These data suggest that radiation-induced alterations in liver nonparenchymal cell populations may be responsible for microvascular fibrosis, which results in a cascade of pathological events which lead to hepatocyte loss.

Vasopressin and A23187 stimulate phosphorylation of myosin light chain-1 in isolated rat hepatocytes

Vasopressin (VP) and other hormones that elevate intracellular Ca2+ concentration also increase tight junctional permeability in the liver cell. Data derived from study of other tissues suggest that microfilaments are instrumental in regulating tight junctional permeability. By analogy to microfilament contraction in smooth muscles, it is likely that the transduction pathway for these hormones involves Ca(2+)-stimulated complex formation between calmodulin and myosin light chain (MLC) kinase with activation of this latter enzyme. MLC kinase then phosphorylates MLC, which, in the presence of actin, exerts adenosinetriphosphatase activity and produces microfilament contraction. This transduction pathway in the hepatocyte remains speculative. To demonstrate the likelihood of this pathway, we stimulated isolated hepatocytes with 10(-8) M VP and assayed MLC phosphorylation. We did this by immunoprecipitation of myosin from homogenates of liver cells prelabeled with [32P]-orthophosphate. We used a polyclonal antibody raised in rabbits against rat liver cell myosin. Our data demonstrate that VP is a potent stimulator of MLC phosphorylation. Maximal rises in intracellular Ca2+ and maximal MLC phosphorylation occur within 40 s of VP administration. The dose-response curve for MLC phosphorylation by VP is similar to that for tight junctional permeabilization in perfused liver with maximal effect at about 10(-8) M VP. The calcium ionophore A23187 also stimulated MLC phosphorylation. MLC phosphorylation, therefore, is at least coincident with, and probably responsible for, tight junctional permeabilization caused by elevation of intracellular Ca2+ in the liver cell.

Hepatic metabolism of artemisinin drugs—II. Metabolism of arteether in rat liver cytosol

1. In this communication, in vitro metabolism of a semisynthetic antimalarial drug arteether in rat liver cytosol is reported. 2. Whenever 14C-labeled arteether was mixed with rat liver cytosol, a crude postmicrosomal fraction of liver cell homogenates, an appearance of three major 14C-labeled metabolites was always attested: deoxy-dihydroartemisinin, AEM-1 (Baker et al., 1988) and metabolite MW286. 3. Transformation of arteether into deoxyDQHS was catalyzed by an enzyme present in the rat liver cytosol, whose activity depended on the presence of NAD +/NADH and a low molecular, dialyzable factor present in the cytosol. The maximal activity of this enzyme was 0.31 nmol of deoxyDQHS formed/min/mg of cytosolic protein. 4. AEM-1 and metabolite mol. wt 286 have been formed directly from arteether by a chemical interaction of the drug with the cytosolic fraction, probably in a non-enzymatic reaction. 5. Taking together the in vitro data of arteether metabolism in rat liver cytosol, presented in this communication, and in vitro data in rat liver microsomes, presented in the preceding communication (Leskovac and Theoharides, 1991), we were able to postulate an integral pathway of Phase I metabolism of arteether in a whole rat liver cell.

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates.

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucokinase is not the pancreatic B-cell glucoreceptor.

A series of recent experimental findings are reviewed to indicate that glucokinase does not represent the pancreatic B-cell glucoreceptor. Whether in liver, pancreatic islet or insulin-producing tumoral cell homogenates, glucokinase fails to yield a higher reaction velocity with alpha-than beta-D-glucose. At a high glucose concentration (40 mmol/l), when the phosphorylation of glucose by glucokinase is indeed higher with beta- than alpha-D-glucose, no preference for beta-D-glucose is observed in intact islets, as judged from the utilization of D-[5-3H]glucose, production of lactic acid, oxidation of D-[U-14C]glucose, net uptake of 45Ca or release of insulin. The glucose 6-phosphate content of intact islets is higher in the presence of beta- than alpha-D-glucose. At a low glucose concentration (3.3 mmol/l), when the participation of glucokinase to hexose phosphorylation is minimal, alpha-D-glucose is still better metabolized and stimulates both 45Ca net uptake and insulin release more efficiently than beta-D-glucose, despite the fact that hexokinase yields a higher reaction velocity with beta- than alpha-D-glucose. In intact islets, beta-D-glucose is used preferentially to alpha-D-glucose in the pentose cycle pathway as judged from the oxidation of alpha- or beta-D-[1-14C]glucose relative to that of alpha- or beta-D-[6-14C]glucose. In islets removed from fasted rats, the rate of glycolysis is more severely decreased than expected from the repression of glucokinase. The metabolism of glucose in tumoral insulin-producing cells differs, in several respects, from that in normal pancreatic islets, although the pattern of hexokinase and glucokinase activities is similar in these two types of cells. All these observations point to the participation of regulatory sites distal to glucose phosphorylation in the control of glucose metabolism in islet cells.

Carbamyl phosphate glucose transferase activity of glucose-6-phosphatase in the isolated rat hepatocyte

Glucose-6-phosphatase (G-6-Pase) will phosphorylate glucose using many compounds as phosphate donors. This activity has been manifested when microsomes are disrupted by exposure to detergent, high pH, or other similar treatment. Others have suggested that this reaction has physiological significance and of the possible phosphoryl donors, carbamyl phosphate is the most likely to be involved in the cell. In our study, rates of glucose phosphorylation were determined in isolated intact hepatocytes incubated under conditions where intracellular CP formation was favored (glucose plus NH 4Cl) or diminished (glucose, NH 4Cl, and ornithine, with and without malate). Results showed that when CP formation was favored, less glucose was phosphorylated than under control conditions (glucose alone). When CP was diminished by the addition of ornithine or ornithine plus malate, no decrease in glucose phosphorylation occurred. The lack of a decrease under these conditions shows that NH 4Cl addition did not mask a stimulation of phosphorylation by CP. Further studies with homogenates of liver cells isolated from these animals did not show an inhibition of glucokinase by NH 4Cl or CP. Thus we have shown that when CP formation was stimulated, glucose phosphorylation was not. If CPGT activity were important in the cell, then under these conditions this should be observed. Since it was not, we conclude that the carbamyl phosphate glucose transferase (CPGT) activity of G-6-Pase is unlikely to have physiological significance.

Analysis of a sheep anti-pig T lymphoblast serum with specificity for E rosette-forming lymphocytes

Sheep antibodies against a pig E-rosette-forming lymphoblastic T lymphoma raised by two intravenous injections of 10 10 cells showed little lymphocytotoxic activity which could be absorbed with red cells, alveolar macrophages or kidney or liver cell homogenates. Bone marrow absorption yielded subpopulation specific antibody which binds to E rosette-forming cells (E.RFC) using either complement-mediated cytotoxicity or indirect antiglobulin rosette formation. In 30 blood lymphocyte preparations from 20 pigs with a range of ∼20–85% E rosettes the mean E% 43.5 ± 2.7 agreed with the % antigen ∗+ cells by cytotoxicity mean = 42.6 ± 2.7 and in each individual sample these figures also agreed closely. In samples of blood lymphocytes enriched and depleted for E rosettes, results of %E ∗+ also agreed closely with % antigen ∗+ cells. This relationship also held for thymocytes and the specific antibodies could be completely absorbed with thymocytes. These data show that the antibody identified peripheral and thymic E.RFC. Bound to lymphocytes the antibody inhibited E rosette-formation with sheep red blood cells (SRBC) in saline (S) and dextran (DS) and with pig RBC in dextran and in Ficoll, but did not affect B cells shown by immunofluorescence, direct antiglobulin rosette formation or Fc rosette-formation, either in saline or dextran, (which include Tγ cells). E rosette inhibition was dependent on antibody concentration, showing single and double sigmoid curves for S and DS rosettes respectively, consistent with differing ease of inhibition of the strong and weak rosette formation. The same spectrum of inhibition of rosette formation by antibody binding followed subsequent incubation with C′ 6-deficient rabbit serum, but with C′-sufficient serum resulted in loss of cells which require dextran for Fc rosette-formation (Tγ). Thus the serum reveals E rosette-forming T cells and their subpopulations, perhaps by binding to the SRBC receptor.

The effect of fatty acid exposure on the biosynthesis of glycerolipids by cultured hepatocytes

Incubation of hepatocyte monolayers with oleate or palmitate (1.0 mM) for 2–48 h, increased (20 to 80%) the incorporation of [1,3- 14C]glycerol and palmitate into triacyglycerol but not phosphatidylcholine. The effect of fatty acids on liver cell triacylglycerol formation correlated well ( r = 0.990) with a simultaneous rise (2–4-fold) in phosphatidate phosphatase (EC 3.1.3.4) activity. Phosphatidate phosphatase activity and triacylglycerol biosynthesis are also increased (2-fold) after hepatocyte monolayers are incubated for 24 h with cyclic GMP in the absence of fatty acids. Fatty acid-dependent increases in liver cell triacylglycerol formation and phosphatidate phosphatase activity are not blocked by cycloheximide. Phosphatidylcholine biosynthesis was also elevated in homogenates of liver cells exposed (24–48 h) to 1.0 mM oleate when exogenous CDPcholine was added to the incubation mixture. Apparently, the phosphatidate phosphatase-dependent rise in diacylglycerols that occurs after fatty acid exposure is primarily shunted into triacylglycerols because liver cell CDPcholine content is not correspondingly increased, and high levels of diacylglycerol acyltransferase (EC 2.3.1.20) and fatty acyl-CoA derivatives are present.

WR-2721, its derivatives and their radioprotective effects on mammalian cells in culture.

Although it is well known that WR-2721 is very efficient in protecting mice against lethal irradiation, we could not find any radioprotective effect of WR-2721 on mouse L cells in culture. But WR-1065 alone (free SH form of WR-2721), and WR-2721 pre-incubated with mouse liver homogenate, showed radioprotective ability. It was found that mouse liver homogenate dephosphorylated WR-2721 to WR-1065. The highest WR-2721 metabolizing activity was found in mouse liver homogenate and Chang liver cell homogenate. Homogenates of human liver and kidney were also shown to possess moderate activity for metabolizing WR-2721. These results suggest to us that WR-2721 must be dephosphorylated before exerting its radioprotective effect and that this dephosphorylating activity varies with tissues. It is demonstrated therefore that mouse L cell in culture is a novel system to assess the extent of dephosphorylation of WR-2721 in various tissues.