Liver Tissue Extracts Research Articles

Study of metabolism of psychotomimetic indolealkylamines by rat tissue extracts using liquid chromatography

The use of a series of liquid Chromatographie techniques involving cation-exchange, reversephase and normal-phase chromatography has permitted the separation and characterisation of a number of metabolites of the psychotomimetic indolealkylamines N, N-dimethyltryptamine and 5-methoxy- N, N-dimethyltryptamine which were isolated following incubation of these compounds with rat tissue extracts. In liver, kidney and brain tissue extracts the routes of metabolism identified included oxidative deamination, N-demethylation, O-demethylation and N-oxidation. The quantitative significance of individual routes of metabolism in these tissues was assessed using N, N-dimethyltryptamine as a substrate.

Site-Finding Behaviour of Fasciola Hepatica (Trematoda), a Parasitic Flatworm

AbstractFasciola hepatica is a trematode parasite of the bile duct of herbivores. Site-finding behaviour of this parasite includes a migration from the duodenum through the abdominal cavity to the liver. This study examines the behaviour of the migrating worms at two stages of the life cycle; NEJ - the stage that penetrates the small intestine, and 48HR - the stage that penetrates the liver. Compared to saline controls, crude extracts of duodenal tissue have greater effects on the behaviour of the NEJs while crude extracts of liver tissue have greater effects on the behaviour of 48HRs. Yet, neither NEJs nor 48HRs migrate up gradients of liver extracts or down gradients of duodenal extracts. Stable chemical gradients are difficult to maintain in the motile abdominal cavity and it is suggested that orientation responses play no part in site-finding behaviour. We propose that the major component of site-finding behaviour of F. hepatica is a passive propulsion of the migrating stages through the viscera and along the body wall to the liver.

Angiotensin-producing serum enzyme II. Formation by inhibitor removal and proenzyme activation.

A highly active angiotensin-producing enzyme (enzyme II) was obtained from dog serum by acid treatment and fractionation to remove angiotensinase and converting enzyme, separate an inhibitor, and convert an inactive precursor (proenzyme II) to enzyme II. Proenzyme II was found to be converted to enzyme II by an endogenous activating enzyme identified as plasmin. Conversion was also caused by the interaction of bacterial streptokinase with human proactivator, by trypsin, and by an activator formed from liver tissue extract and dog serum. Neither plasma kallikrein nor the labile, human extrinsic tissue-type plasminogen activator induced activation. The inhibitor, which normally blocks the activation of proenzyme II, was unusually stable against high temperatures and extremes of pH, and it was not identical to any of the six known protease inhibitors of serum. Enzyme II was not identical to other angiotensin-producing enzymes such as enzyme I, renin, cathepsin D, pepsin, plasmin, tonin, or cathepsin G. Enzyme II reacted maximally at pH 4.7 and produced up to 2250 ng of angiotensin I/ml serum/hr from the substrate of dog serum (i.e., amounts 3200-fold higher than that produced by endogenous renin of normal dog serum). Since at pH 7.2, angiotensin I formation is still about 30 times higher than that of renin, enzyme II may be physiologically active under some conditions.

Insulin degradation: radioimmunoassay for glutathione-insulin transhydrogenase and its application.

A double-antibody radioimmunoassay for the insulin-degrading enzyme, glutathione-insulin transhydrogenase (GIT), has been developed with the use of rabbit antiserum against human liver GIT and [125I]-GIT. The method can determine as little as 32 fmol of GIT, thus allowing measurements in needle tissue biopsy samples and in plasma, which have not been possible with previous enzymatic procedures. Relative competition in the radioimmunoassay by unlabelled GITs purified from other sources are in agreement with homologies in GITs previously found using the enzymatic assay. No competition was observed with pork insulin, bovine ribonuclease, human albumin or human gamma-globulin, indicating that the radioimmunoassay is highly specific for GIT. Similar competition curves were observed for native GIT; active, reduced GIT; or for the inactive, S-(ethylsuccinimido) derivative of GIT. The radioimmunoassay thus measures total (active + inactive) GIT and permits determinations in the presence of materials which react with the active site and render the enzymatic methods unusable. Radioimmunoassay of plasma and extracts of liver, muscle and adipose tissues from diabetic and non-diabetic subjects showed parallel competition curves with standard purified human GIT indicating that GITs of non-diabetic and diabetic persons are immunologically very similar or identical. Concentrations of GIT in plasma determined by radioimmunoassay were significantly higher in diabetic than those in non-diabetic subjects (1620 +/- 80 versus 1070 +/- 30 fmol/l, p less than 0.001). Tissue GIT levels found by the radioimmunoassay as well as by the enzyme assay, both in non-diabetic and diabetic subjects, were highest in the liver, intermediate in the adipose tissue and lowest in the muscle.

Determination of mouse liver 5-methyltetrahydrofolate concentration and polyglutamate forms

The concentration and polyglutamate status of 5-methyltetrahydrofolate in mouse liver tissue extracts has been determined by enzymatic conversion to methylenetetrahydrofolate and subsequent entrapment of this confactor form into a ternary complex with Lactobacillus casei thymidylate synthase and tritiated 5-fluorodexyuridylate. 5-Methyltetrahydrofolate was oxidized to methylenetetrahydrofolate using the reverse reaction of methylenetrahydrofolate reductase with menadione as the ultimate electron acceptor. Reference 5-methylterahydrofolate could be quantitatively recovered from tissue extracts by this method. The polyglutamate status of enzymatically converted and complexed tissue 5-methyltetrahydrofolate was determine electrophoretically. Unlabeled 5-fluorodeoxyuridylate was used to remove endogenous methylenetetrahydrofolate prior to enzymatic oxidation of 5-methyltetrahydrofolate and subsequent electrophoretic analysis. In this manner, the 5-methyltetrahydrofolate polyglutamate pool alone could be labeled and visualized. There were no observable differences in the polyglumate distribution of endogenous methylenetetrahydrofolate versus 5-methyltetrahydrofolate polyglutamates of normal mouse liver tissue.

Modified mutagen metabolism in Schistosoma hematobium-infested organisms.

The relationship between parasite infestation and chemical mutagen metabolism was investigated in this study. Schistosoma hematobium, long associated with increased incidence of bladder cancer in humans, was chosen as a model parasite. Urine samples, serum samples, and liver tissue extracts (S-9) from infested and control hamsters were used with the Ames Salmonella/microsome test to follow 3,3'-dichlorobenzidine (DCB), aflatoxin B1 (AFB1), and 2-acetylaminofluorene (AAF) mutagenicity. Liver S-9 preparations from infested and control hamsters showed little difference in activation potential for DCB and AFB1. Aroclor 1254-induced rat liver S-9, however, was remarkably efficient at reducing the mutagenicity of DCB. This process was reversible by beta-glucuronidase (BG). Studies on infested and control hamsters indicated increased BG activity in serum and urine. Urine concentrates (UC) from infested and control animals were not mutagenic by themselves, but did enhance the mutagenicity of AAF and DCB in the presence of S-9 and BG. Urine concentrates from infested animals showed greater enhancement of DCB mutagenicity than did UC from control animals. These data suggest that increased BG and unknown urinary factors in infested hamsters play a role in altering chemical mutagen activity.

A study on phosphoglucose isomerase enzyme in some elasmobranch fishes collected from khor al-zubair, North west of the Arabian Gulf, Iraq

1. 1. Tissue extracts of skeletal muscle, brain, gills, liver, kidney, heart, eye and spleen were electrophoretically examined for phosphoglucose isomerase (EC 5.3.1.9) activity in five species of elasmobranch fishes. 2. 2. Two phosphoglucose isomerases (PGI) with different specification have been found in all groups of elasmobranch fishes studied. 3. 3. PGI proved to be a good taxonomic criterion to differentiate members of the families Sphyrindae and Dasyatidae from the other remaining elasmobranch families. 4. 4. PGI appeared to be controlled by duplicated genes as in other teleostean fishes.

Isolation and sequence analysis of human bombesin-like peptides

The decapeptide form of human gastrin releasing peptide was isolated from acid extracts of liver tissue containing a metastatic human bronchial carcinoid tumor. A larger form also was isolated and partially characterized. During gel permeation chromatography the major immunoreactive peak eluted in the same region as synthetic gastrin releasing decapeptide while a second minor immunoreactive peak eluted near gastrin releasing peptide. Bombesin-like immunoreactivity (BLI) was purified by successive applications to reverse phase high pressure liquid chromatography (HPLC) columns. After four successive HPLC purifications a single peak of bombesin-like immunoreactivity was detected. Amino acid analysis, microsequence analysis and coelution with synthetic peptide indicated that the predominant form present in metastatic tumor tissue was identical to the decapeptide form of canine gastrin-releasing peptide. The less abundant form was purified by cation exchange chromatography followed by reverse phase high pressure liquid chromatography. Partial microsequence analysis of this peptide, through the first 11 residues, was Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr-Val-Leu. This sequence differed from that of hog heptacosapeptide gastrin releasing peptide at positions 1,3,4 and 5 and from the canine peptide as positions 1,3,5, and 7.

The concentration of P-enolpyruvate carboxykinase protein in murine tissues in diabetes of chemical and genetic origin.

P-enolpyruvate carboxykinase protein was measured by radioimmunoassay in liver, kidney, and adipose tissue extracts from alloxan- and streptozotocin-diabetic rats, in liver extracts from C57BL/KsJ-db+/db+ "diabetic" mice and in liver extracts from normal mice subjected to different dietary or hormonal states. The radioimmunoassay method measured tissue enzyme concentration (nanomoles/g) and total organ enzyme content (nanomoles/liver) independently of assayable activity (units/g). The "apparent" specific activity (units/nmol) was calculated from the maximum velocity and enzyme concentration data. Extracts of rat liver mitochondria and of skeletal muscle cytosol were also analyzed for P-enolpyruvate carboxykinase by the radioimmunoassay. In "chemical" diabetes, P-enolpyruvate carboxykinase by the radioimmunoassay. In "chemical" diabetes, P-enolpyruvate carboxykinase concentration increased approximately 3-fold over the fed, normal liver value of 0.89 microM, approximately 1.6-fold over the normal kidney value of 1.9 microM and approximately 2.9-fold over the normal adipose tissue value of 0.030 microM. Chemical diabetes caused the specific activity to decrease from 0.38 to approximately 0.27 units/nmol in liver and from 0.48 to approximately 0.32 units/nmol in kidney. Insulin replacement by in vivo injection not only promptly lowered the abnormally high enzyme concentration in both tissues but, paradoxically, decreased the apparent specific activities further to approximately 0.16 in liver and to 0.23 in kidney. In the db+/db+ diabetic mouse the liver enzyme increased from 0.22 microM at 5 weeks of age to 0.44 microM at 18 weeks when serum glucagon concentration is known to be highest and the pancreatic beta cells to be depleted of insulin. While the enzyme protein concentration increased 2-fold in the 18-week-old db+/db+ mouse, total liver enzyme content had actually increased 5-fold due to liver enlargement. In fasted normal mice, glucagon-treated normal mice, and alloxan/streptozotocin-treated mice, the concentration of liver enzyme increased significantly compared to the values in fed control mice. Pharmacological doses of dexamethasone did not induce the mouse enzyme. Rat liver mitochondria contained only trace quantities of immunoassayable enzyme which can be explained by contamination with cytosolic proteins. Rat skeletal muscle also contained only insignificant quantities of the enzyme or perhaps another cross-reacting, immunoassayable protein. The data obtained in diabetes before and after treatment show that complex mechanisms of control by insulin and glucagon do operate to regulate P-enolpyruvate carboxykinase in liver, kidney, and adipose tissue.

Erythropoietin in liver tissue extracts and in liver perfusates from hypoxic rats

An attempt to evaluate the role of the liver in extrarenal erythropoietin production was made by measuring the content of erythropoietin in homogenates and perfusates from hypoxic rat livers. Extracts from livers from nephric or anephric animals rendered both anemic and hypoxic showed no detectable erythropoietin despite the fact that both plasma and kidney extracts contained large amounts of erythropoietin. This lack of measurable erythropoietin in the liver is not caused by degradation of erythropoietin during the extraction procedure because exogenously added rat erythropoietin was recovered to the same extent from livers or kidney homogenates. More likely, however, it is caused by the fact that extrarenal erythropoietin production accounts for only one-fifth of total erythropoietin production and that the liver mass is about six times that of both kidneys. Consequently, the erythropoietin content of 1 g of liver should be about one-thirtieth of that of 1 g of kidney, an amount that is below the limit of detection of the assay. On the other hand, the 2-h in situ perfusates of livers from similarly stimulated animals contained significant amounts of secreted erythropoietin. It is concluded that the liver participates actively in extrarenal erythropoietin production in the adult rat. However, the small amount expected to be present in tissue homogenates cannot be detected with our current bioassay.

Experimental infection of young rabbits with rabbit parvovirus.

A newly isolated parvovirus of rabbits (F-7-9 strain) was inoculated into young female rabbits by the oral or intravenous route. Virus was recovered from the feces 3 to 13 days after inoculation. Viremia was demonstrated in orally inoculated rabbits. The virus was also isolated from tissue extracts of liver, pancreas, spleen, small intestine, appendix and mesenteric lymph node on days 10 and/or 14, and from the small intestine on day 30 after inoculation. Hemagglutination inhibiting antibody appeared in the serum after 8 days and neutralizing antibody was detected later. An 11S neutralizing antibody was demonstrated in fecal extracts between 15 and 30 days after inoculation. Infected rabbits showed very mild clinical signs of listlessness and inappetence lasting for a few days. Histologically, mild to moderate catarrhal enteritis was observed in the small intestine.

Immunological characterization of cytochrome b9 in crude extracts from rat tissues.

Rabbit antiserum against rat liver-soluble cytochrome b9 was produced. The antiserum was specific to cytochrome b9 and did not react with hemoglobin, cytochrome c, cytochrome H-450, serum, hemolysate, and tissue extracts from lung, brain, testis, and skeletal muscle. From Ouchterlony double immunodiffusion and tandem crossed immunoelectrophoresis, the antigen was found in liver, kidney, and heart tissue extracts of rat with complete identity. From crossed immunoelectrophoresis, the antiserum distinguished monomeric and dimeric forms of the antigen. Rocket immunoelectrophoresis for quantitating the antigen was developed with a minimum sensitivity of 5 ng. By using rocket immunoelectrophoresis, the concentration of the antigen in crude tissue extracts from liver, heart, and kidney was determined. The antigen was not detectable in crude extracts from lung, brain, testis, and skeletal muscle by immunological methods. Therefore, the immunological assay method was very useful for identifying and quantitating cytochrome b9 in crude extracts with high sensitivity.

Function of hepatic triglyceride lipase in lipoprotein metabolism

Rat hepatic triglyceride lipase (H-TGL) was purified from liver tissue extracts by affinity chromatography on Sepharose 4B with covalently linked heparin. The purified rat H-TGL exhibited the properties previously described for this enzyme. Enzyme protein was injected into rabbits for anti-H-TGL antibody production. Antirat-H-TGL did not react against rat lipoprotein lipase (LPL) but inhibited H-TGL-activity both in vitro and in vivo greater than 90%. These antibodies were injected into rats and lipoprotein analyses were performed over a 36-hr period. It could be shown that inactivation of H-TGL by anti-H-TGL gamma-globulins in vivo led to an increase in total triglyceride concentration from 70 mg/dl to 230 mg/dl due to an increase in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) triglycerides 4 hr after antibody injection; a marked increase in high density lipoprotein (HDL) phospholipid concentration was observed with almost no change in HDL-cholesterol and HDL-triglycerides. This study documents the ability of antirat-H-TGL-gamma-globulins to inhibit H-TGL in vitro and in vivo. Furthermore, the inhibition of triglyceride removal in vivo demonstrated that this enzyme together with LPL is responsible for the catabolism of VLDL-triglyceride.

Tissue distribution, excretion, and metabolism of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in the Golden Syrian hamster

The hamster has been reported to be the least sensitive mammalian species to the acute toxicity of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The fate of a single dose of [ 3H]- or [ 14C]TCDD (650 μg/kg, ip or po) was assessed in male hamsters for up to 35 days following treatment. The greatest content (percentage dose/g tissue) of radioactivity was found in the liver, adipose tissue, and adrenals. The radioactivity in liver and adipose tissue was identified as unmetabolized TCDD. The rate of 3H or 14C elimination in urine and feces suggested a first-order process. Similar half-life of elimination ( t 1 2 ) values of 12.0 ± 2.0 and 10.8 ± 2.4 days (mean ± SD) were obtianed with ip administered [ 3H]- and [ 14C]TCDD, respectively. With both [ 3H]- and [ 14C]TCDD, approximately 35 and 50% of the radioactivity was eliminated in urine and feces, respectively. The t 1 2 for po administered [ 3H]TCDD was 15.0 ± 2.5 days. High-pressure liquid chromatography of the urine and bile of animals receiving [ 14C]TCDD revealed one major and several minor radioactive peaks, none of which corresponded to [ 14C]TCDD. The apparent absence of TCDD metabolites in extracts of liver or adipose tissue indicates that the biotransformed products of TCDD are readily excreted in urine and bile. The enhanced rate of metabolism and excretion of TCDD in hamsters relative to other species may in part contribute to, but not totally explain its unusual resistance to TCDD toxicity.

Tritiated 17β-estradiol uptake by the brain and other tissues of the cichlid jewel fish, Hemichromis bimaculatus

Intact and gonadectomized mature and immature Hemichromis bimaculatus were treated with tritium-labeled estradiol to investigate estrogen uptake by various tissues. The metabolism of the injected estradiol was examined by column chromatography of blood, liver, kidney, optic tract, and brain tissue extracts. The inferior lobe of the brain (hypothalamus and hypophysis) showed a characteristic pattern of uptake and retention of radioactivity similar to that shown by other vertebrates in that the uptake was not displaced by a second injection of unlabeled estrogen but could be blocked by preinjection of the unlabeled steroid. Chromatographic analysis of tissue extracts indicated the bound hormone to be predominantly 17β-estradiol. Regions of the brain and other body tissues which did not differentially take up and retain radioactivity contained a relatively larger fraction of the label as estrone or more polar compounds. The cerebrum, although containing less radioactivity as estradiol than the inferior lobe, did retain a greater percentage of its radioactivity as estradiol than the remaining brain regions.

Simple radioassay for uridine phosphorylase and 5′-nucleotidase

A simple micromethod was developed for the accurate measurement of the activity of uridine phosphorylase in the direction of both ribosylation and phosphorolysis. The technique was applicable also to the measurement of cytoplasmic 5′-nucleotidase. In this micromethod the radioactive products were separated from the substrates by electrophoresis on cellulose acetate membrane in 0.1 m borate buffer, pH 9.0, at 150 V for 40 min. These micromethods are highly sensitive and capable of utilizing small samples (4–30 μg protein); they are valid in crude tissue extracts of rat liver and hepatoma.

Evidence that somatomedin is synthesized by multiple tissues in the fetus

Production of somatomedin-C, a growth hormone-dependent peptide believed to mediate the growth-promoting actions of growth hormone, has been assessed using explants of fetal mouse tissues. Quantitation of this peptide in media of explants cultured for 3 days has been accomplished with a membrane receptor assay for somatomedin and a specific radioimmunoassay for somatomedin-C. Somatomedin-C is produced by the 11-day-gestation fetal mouse liver, increases exponentially in parallel with liver growth until the 16th day of gestation, and falls postnatally. Media somatomedin is believed to be derived by de novo synthesis since saline extracts of liver and most other fetal tissues contain only a small fraction of the activity in culture media. The immunoreactive material secreted into media appears to be closely related to human somatomedin-C since it produces dilution curves which are parallel to those of pure hormone, migrates on Sephacryl 200 at a size similar to that of one of the components of human serum somatomedin-C, dissociates into small molecular weight material with acid treatment, and isofocuses in a range comparable with that of somatomedin-C purified from human serum. Eleven-day limb bud mesenchymal micromass cultures and 17-day-gestation intestine, heart, brain, kidney, and lung also synthesize immunoreactive somatomedin-C in serum-free medium. For these tissues, the media activity was far in excess of the tissue extractable activity. Somatomedin activity in excess of the tissue extractable activity, however, was not found in media from 17-day-gestation placenta. The finding that multiple tissues synthesize somatomedin-C raises the possibility that the primary biological actions of this hormone are exerted locally at its sites of origin. Although a function of this type by a peptide has not been widely suspected, it seems plausible that the cells of fetal tissues are capable of producing local mitogens in much the same manner as the postulated inducers of tissue differentiation.

The presence of a low molecular weight acid phosphatase in liver tissue that cannot be demonstrated with the histochemical substrate naphthol AS-BI phosphate.

Three distinct isoenzymes of acid phosphatase have been separated from extracts of liver tissue of rats by gel filtration. These isoenzymes have molecular weights of 180,000±35,000; 74,000±11,000 and 13,000±2,500. High molecular weight isoenzymes and a low molecular weight isoenzyme of acid phosphatase (molecular weight 13,000±2,100) were also present in extracts of normal human and mouse liver tissue, and of pathologically altered liver tissue of mice in which the activity of acid phosphatase was strongly increased as a result of intraperitoneal injections of dextran solutions. Activity of acid phosphatase was determined with three substrates. The isoenzymes showed different conversion rates for the three substrates. The high molecular weight isoenzymes split the substrates 4-methylumbelliferyl phosphate, p-nitrophenyl phosphate and naphthol AS-BI phosphate. The activity was sensitive to the inhibitors fluoride and L(+)tartrate. In the pathologically altered liver tissue, which had stored dextran, the activity of these isoenzymes was strongly increased. The low molecular weight isoenzyme split 4-methylumbelliferyl phosphate and p-nitrophenyl phosphate but not naphthol AS-BI phosphate. Therefore this isoenzyme cannot be demonstrated with histochemical techniques using the substrate naphthol AS-BI phosphate. In contrast to the activity of the high molecular isoenzymes the activity of the low molecular isoenzyme was not changed in the pathologically altered liver tissue of mice and was not sensitive to the inhibitors fluoride and L(+)tartrate.

A selective deficiency of hepatic triacylglycerol lipase in guinea pigs

The properties of postheparin plasma triacylglycerol-hydrolyzing enzymes were investigated in guinea pig and rat. In rat, lipoprotein lipase and hepatic triacylglycerol lipase were separated on a heparin-Sepharose affinity chromatography. In postheparin plasma of guinea pig, however, hepatic triacylglycerol lipase was almost completely absent, while lipoprotein lipase was present. Hepatic triacylglycerol lipase was also deficient in the liver tissue extract of guinea pig. Plasma lipoprotein compositions of high-fat fed and control guinea pigs were analyzed. One of the outstanding changes found in high-fat fed animals was the presence of chylomicronemia. One guinea pig showed gross hyperlipemia with triacylglycerol concentrations of 2715 mg/100 ml. Plasma triacylglycerol concentrations of each lipoprotein fraction of very low density, intermediate density, low density and high density lipoproteins from high-fat fed animals were almost the same as those of the corresponding lipoprotein fractions from controls. Discussion was focused on the development of chylomicronemia in relation to the defects of triacylglycerol-hydrolyzing enzyme systems in this animal.

Immunological cross-reactivity between acid extracts of myelin, liver and neoplastic tissues: studies in immunized guinea-pigs.

Groups of 4 guinea-pigs were immunized with acid extracts prepared from bovine myelin (EF), normal human liver tissue and malignant or benign neoplastic tissues in Freund's complete adjuvant (FCA1. The animals were weighed daily and examined for clinical signs of experimental allergic encephalomyelitis (EAE). All the animals immunized with EF developed clinical symptoms of EAE within 21 days of the initial immunization, whilst some of the animals immunized with certain tumour extracts developed symptoms which closely resembled those of EAE. Control animals immunized with FCA only remained asymptomatic. Cellular immunity to the various extracts in immunized animals was assessed 20 days after immunization by i.d. skin testing, and upon killing at Day 21 with the direct peritoneal-exudate macrophage migration inhibition (MMI) test. Brains and spinal cords were removed at killing, fixed in formalin and processed for histological examination. I.d. skin testing was shown to be most consistent in demonstrating positive delayed hypersensitivity, whilst the MMI test frequently gave negative results in the presence of pronounced skin responses to specific extracts. Thus it was shown that 3/4 animals immunized with basic proteins extracted from an adenocarcinoma of the lung or related hepatic metastases, and 1/2 animals immunized with an extract of a carcinoma of the breast, gave intense erythema and induration responses 5 mm in diameter 24 h after i.d. challenge with EF. No such response was obtained in animals immunized with basic proteins extracted from normal human liver, any of the other neoplastic tissues, or in control animals immunized with FCA only. Examination of brains and spinal cords from animals immunized with EF revealed dense infiltration by mononuclear cells in the ependyma and choroid plexus of levels in the spinal cord. Examination of brains and spinal cords from animals immunized with the lung-tumour extract or related hepatic metastases which showed demonstrable immunological cross-reactivity with EF in immunized animals, revealed a number of inflammatory changes characterized by dense infiltrates of mononuclear cells sub-ependymally, and perivascular cuffing in the cortex. However, no significant lesions were seen in the spinal cords of these animals. Polyacrylamide-gel electrophoresis of the 2 tumour extracts exerting this apparent encephalitogenic effect did not reveal proteins within the mol. wt range of EF. Thus the observed pathological effects and cross-reactivity with EF were probably not due to contamination with nervous-tissue components. It is suggested that these tumour extracts may have contained a component or components other than EF, immunologically cross-reactive with EF, and capable of inducing the observed encephalitis.