Mitochondrial swelling and microtubule depolymerization are associated with energy depletion in axon degeneration.

Although mitochondrial dysfunction is intimately related to axonal degeneration following nerve injury, the molecular mechanisms of mitochondrial swelling and its mechanistic relation to axonal degeneration are largely unknown. Previous studies have demonstrated that axonal degeneration in the injured peripheral nerves shows two morphologically distinct phases: (1) A latency period (∼24h), in which the morphology of axonal cytoskeletons seems unchanged, followed by (2) an execution period (36-48h), which shows a catastrophic granular degeneration of most axonal structures in rodent axons. In the present study, we found that, in the sciatic nerve axotomy model, energy failure and microtubule depolymerization occurred during the latency period whereas mitochondrial swelling and neurofilament degradation started in the execution period. The energy repletion with NAD or an NAD/pyruvate mixture inhibited microtubule depolymerization, mitochondrial swelling and axonal degeneration in transected sciatic nerve axons. In addition, microtubule perturbing agents enhanced axonal degeneration and mitochondrial swelling. Extracellular calcium chelation did not affect energy failure, microtubule depolymerization or mitochondrial swelling, but it did prevent neurofilament degradation. These findings suggest that an early disturbance in energy dynamics regardless of mitochondrial swelling might be a key trigger for the initiation of axonal degeneration and that extracellular calcium influx is a late effector for neurofilament degradation.

Expression of Nov, CYR61 and CTGF genes in human hepatocellular carcinoma.

CCN family genes, Nov, CYR61 and CTGF, are immediate-early genes expressed in fibroblasts following growth stimuli. Aberrant expression of Nov has been found in human Wilms' tumor, and suggested to be involved in tumorigenesis. The aim of our experiments is to examine the expression of CCN family genes in human hepatocellular carcinomas (HCCs) and find the correlation of these gene expressions with clinicopathological parameters. A pair of tumor and surrounding non-tumor tissues were obtained from 23 patients with HCC and six with metastatic liver tumor. Total cellular RNA isolated from tissues was analyzed for the presence of mRNA of CCN family genes by the reverse-transcription polymerase chain reaction. Nov, CYR61 and CTGF mRNA were identified in 17 (73.9%), 17 (73.9%), six (26.1%) tumors, and in nine (39.1%), 16 (69.6%), one (4.3%) surrounding non-tumor tissues of 23 patients with HCC. No significant difference was found in clinicopathological parameters between cases with HCC negative and positive for these gene expressions. The prevalence of Nov and CTGF expression in HCC is significantly higher than those in surrounding non-tumor. The same tendency was found in metastatic tumors. These results suggest that Nov and CTGF is associated with the development of tumors in the liver.

Kinetics of expression of connective tissue growth factor gene during liver regeneration after partial hepatectomy and D-galactosamine-induced liver injury in rats.

Connective tissue growth factor (CTGF) is up-regulated by TGF-beta1 during wound healing. The present study examined the expression of CTGF during regeneration after 70% partial hepatectomy (PH) or d-galactosamine (GalN)-injured liver in rats. CTGF, TGF-beta1, and type I collagen mRNAs were semiquantified by a ribonuclease protection assay. After PH, TGF-beta1 and type I collagen were increased at 2-6 h and at 12-48 h. CTGF increased at 6 h and returned to the control level thereafter. The ribonuclease protection assay of cultured hepatic stellate cells (HSC) and in situ hybridization suggest that the cells express CTGF along sinusoid might be HSCs. After GalN administration, CTGF increased at 2-96 h with a shoulder peak at 6-12 h followed by a main peak at 24 h. TGF-beta1 and type I collagen were up-regulated with kinetics similar to those of CTGF. The different kinetics between PH and GalN regenerations indicate that regulation of CTGF in the two processes is different. Higher TGF-beta1 expression after inflammatory/necrotic process in the GalN regeneration may caused the prolonged CTGF expression.

Replication of hepatitis B virus which carries foreign DNA in vitro.

Targeting a specific DNA sequence to the desired tissues is an important step in gene therapy. The hepatitis B virus (HBV) is the only DNA virus that has hepatocyte specificity. We attempted to construct an HBV-based vector for targeting the liver. We observed the replication and secretion of virus particles in an HBV construct that lacks X gene and carries an extra 63 bp DNA fragment in vitro. Replication was observed in the cell line HuH-7 but not HepG2. From this construct, we designed an HBV-based vector that could carry foreign DNA. HBV based vectors provide for the possibilities of generating therapeutic agents for individual patients. Our host vector system may be used to clear out the HBV from the HBV carrier or chronic hepatitis B patients by introducing a genetically engineered HBV into these patients.

Tuberous sclerosis associated with multiple hepatic lipomatous tumors and hemorrhagic renal angiomyolipoma.

We report a case of tuberous sclerosis associated with hepatic lipomatous tumors and renal angiomyolipomas. Abdominal ultrasonography revealed a high echoic large tumor in the left kidney. A provisional diagnosis of angiomyolipomas of the kidney was made based on computed tomography. Subsequent laparotomy revealed that the extracted tumor was renal angiomyolipoma. It was also revealed that there was an association with hepatic lipomatous tumors thought to be lipomas or angiomyolipomas by liver biopsy. Nearly half of all cases of angiomyolipoma in the kidney are reported as occasional association with tuberous sclerosis complex, but lipomatous tumors in the liver are rare.

Expression of the midkine gene in human hepatocellular carcinomas.

BACKGROUND/AIMS: Aberrant expression of Midkine (MK) has been found in various human carcinomas including hepatocellular carcinoma (HCC). The aim of study is to identify the incidence of MK expression in tumor and surrounding non-tumor tissues of the liver, and to find the correlation of MK expression with other tumor markers. METHODOLOGY: Liver tissues were obtained from 16 patients with HCC and 4 with metastatic liver cancer. Background diseases of the HCC patients include liver cirrhosis and chronic hepatitis of type B or C. RNA was prepared from both cancerous and surrounding non-cancerous tissues, and analyzed for the presence of MK mRNA by RT-PCR, PCR-Southern blot, and Northern blot analysis. RESULTS: MK expression was detected in 12 (75%) of 16 HCCs by PCR-Southern blot analysis, the most sensitive of the 3 methods. Three of 9 surrounding cirrhotic tissues were weakly positive for MK expression, and none of chronic hepatitis and 4 normal tissues were negative. No significant difference was found in clinical and pathological parameters between MK negative and positive cases. Among metastatic cancers, 1 of gastric origin was positive for MK expression, but 1 each of chorangiocellular, gall bladder, and gastrinoma origin was negative. CONCLUSIONS: These results suggest that MK is expressed in the majority of HCC tissues and rarely in surrounding tissues in chronic liver diseases.

Inter-laboratory difference among eleven clinical laboratories in the Okayama City area.

The aim of the present study was to find the cause of inter-laboratory differences in laboratory test data and to examine whether control assessment helps to reduce inter-laboratory differences. Blood and serum samples of one healthy subject and one subject with liver cirrhosis were analyzed by 11 laboratories in the Okayama City area. No differences were found in the assay units of 26 tests surveyed. However, considerable differences were observed in test data, reference interval, and clinical level (CL), though most laboratories pointed out that the test data for the normal subject was within the reference intervals and those for the patient with liver cirrhosis showed abnormalities in tests for liver function. The difference in reference intervals was serious in the tests of direct bilirubin (D-Bil), thymol turbidity test (TTT), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (GGTP) and choline sterase. Marked differences in CLs were found in the tests of D-Bil, TTT, ALP, GGTP, creatine phosphokinase, amylase, heavy density lipoprotein cholesterol and white blood cell count. However, three hepatologists independently suggested that such inter-laboratory differences would not seriously affect a clinical decision on the disease status of the cirrhotic patient. Most tests that showed a trend error in a recent quality control survey appeared to have the same trend in the present study. These results indicate that inter-laboratory differences occur at various levels and control assessment are helpful in establishing, and therefore reducing, the level of inter-laboratory differences.

Increase in the expression of biglycan mRNA expression Co-localized closely with that of type I collagen mRNA in the infarct zone after experimentally-induced myocardial infarction in rats.

Biglycan, a small dermatan sulphate proteoglycan, has been postulated to interact with other components of the extracellular matrix (ECM), specifically collagens. We hypothesized that biglycan messenger ribonucleic acid (mRNA) is increased in the myocardial infarct zone. Biglycan mRNA expression after acute myocardial infarction (AMI) in rats was determined with the use of Northern blotting and in situ hybridization, and its expression pattern was compared to that of type I collagen mRNA [alpha1(I) collagen]. The left coronary artery was ligated in male Sprague-Dawley rats, and the hearts were excised on days 2 and 7. The Northern blot analysis demonstrated that expression of biglycan mRNA in the infarct on days 2 and 7 were 4.0- and 6.8-fold higher, respectively, compared to the sham-operated hearts. The in situ hybridization revealed intense signals for both biglycan and alpha1(I) collagen mRNA on day 2 in the spindle-shaped mesenchymal cells located between the surviving myocytes in the infarct peripheral zone. On day 7, biglycan mRNA signals were observed in the interior of the infarct around the infarct granulation tissue, a distribution that was essentially the same as that of alpha1(I) collagen. These results demonstrated that the increases in the infarct biglycan mRNA expression produced by mesenchymal cells (presumably myofibroblasts and fibroblasts) was closely co-localized with that of type I collagen mRNA, indicating that biglycan contributes to the infarct healing processes.

Point mutations in the S and pre-S2 genes observed in two hepatitis B virus carriers positive for antibody to hepatitis B surface antigen.

Two hepatitis B virus (HBV) carriers who had antibodies to HBV surface antigen (anti-HBs) were studied. Case 1 was a 47 year old woman positive for hepatitis B e antigen (HBeAg), and case 2 was a 61 year old man positive for antibody to HBeAg (anti-HBe) and DNA-polymerase (DNA-p). Neither case had received the HBV vaccine. The nucleotide sequences of the HBV-DNA extracted from the patients' sera were determined within the pre-S2 and S genes. Seven out of nine S gene clones from case 1 and six out of nine S gene clones from case 2 had an amino acid replacement from Thr or Ile to Ser at codon 126 in the alpha-determinant of the S gene. Amino acid substitution of codon 145 of the S gene previously reported was not observed. Although two previous reports on HBV escape mutant carriers with both anti-HBs and HBeAg described some deletions in the pre-S2 gene, our cases did not show these deletions. Our analysis indicated that carriers with the HBV escape mutant did not always have pre-S2 gene deletions. We found two HBV escape mutant carriers who had amino acid substitutions at codon 126 in the S gene due to point mutation without any deletions in the pre-S2 gene.

Time-dependent increases in syndecan-1 and fibroglycan messenger RNA expression in the infarct zone after experimentally induced myocardial infarction in rats.

BACKGROUND: Syndecan-1 and fibroglycan, heparan sulphate proteoglycans, play important roles in extracellular matrix formation via their biological functions. OBJECTIVE: To examine experimentally the sequential changes in syndecan-1 and fibroglycan messenger RNA (mRNA) expression after acute myocardial infarction. MATERIALS AND METHODS: The left coronary arteries of male Sprague-Dawley rats were ligated and the hearts were excised on days 1-14, 28 and 42. Syndecan-1 and fibroglycan mRNA expression in the infarct and non-infarct zones and in sham-operated hearts was determined by reverse transcriptase-polymerase chain reaction. Amplified products were quantified by densitometry of the electrophoresed bands stained with ethidium bromide and standardized relative to the glyceraldehyde 3-phosphate dehydrogenase or beta-actin mRNA expression. Northern hybridization was also performed in the infarct and non-infarct zones on day 3. RESULTS: Expression both of syndecan-1 and of fibroglycan mRNA began to increase on day 2. The expression attained maximum levels on day 3. The maximum levels of syndecan-1 and fibroglycan expression were, respectively, sevenfold and fivefold the preligation level and the level in the sham-operated hearts. The levels remained elevated until day 14, whereupon they declined gradually, returning to the control levels by around day 42. Northern blotting also demonstrated that there was an increased expression both of syndecan-1 and of fibroglycan mRNA in the infarct compared with that in the non-infarct zone on day 3. CONCLUSION: Our results demonstrated that there are sequential increases in the expression both of syndecan-1 and of fibroglycan mRNA in the infarct zone after experimentally induced myocardial infarction in rats, suggesting that these proteoglycans play some role in the pathological course of infarction.

Reciprocal gene expression of rat fibroglycan and beta-actin during the course of regeneration after D-galactosamine liver injury.

BACKGROUND/AIMS: Fibroglycan (FG) is a major heparan sulfate proteoglycan (HSPG) in the rat liver that is mainly distributed on the surface of hepatocytes. HSPG may play some important roles in the regeneration of liver by interacting with various growth factors such as bFGF and HB-EGF. However, little is known about the function of FG. We reported that after injury caused by D-galactosamine, regeneration started on the following day and peaked on day 2. To clarify the function of FG in liver regeneration, we investigated the gene expression of FG during regeneration after D-galactosamine injury. MATERIALS AND METHODS: Rats were given D-galactosamine on day 0. Liver RNA was collected from day 0 to day 7. The gene expression of FG and beta-actin (as a representative cytoskeleton) was examined by Northern and/or Slot blotting. RESULTS: FG gene expression was markedly decreased on day 2, but totally recovered on day 3. In contrast, beta-actin gene expression was markedly increased on day 2 and returned to the normal level on day 3. Expression of the FG and beta-actin genes was reciprocal. CONCLUSION: FG expression is transiently suppressed when cytoskeleton gene expression is enhanced at the early phase of liver regeneration.

Promoter-independent loss of mRNA and protein of the Rb gene in a human hepatocellular carcinoma.

BACKGROUND: Inactivation of the retinoblastoma (Rb) gene is considered to play a fundamental role in the genesis and progression of several human cancers. In retinoblastoma, the inactivation of Rb promoter by mutations or hypermethylation has been reported. Although genetic changes of Rb gene have been described in hepatocellular carcinoma (HCC), an epigenetic change such as hypermethylation of the Rb promoter as reported in retinoblastoma has not been described. MATERIALS AND METHODS: We examined the hypermethylation in the promoter region of Rb gene by restriction fragment length polymorphism in 19 HCCs, as well as the expression of Rb mRNA and protein by RT-PCR and by immunoblotting, respectively. RESULTS: We found no evidence of hypermethylation in the promoter region of the Rb gene in all HCCs analyzed. However, the expression of Rb mRNA and protein was lost in one HCC, and no mutation was detected in the Rb promoter region of this patient. The inactivation of Rb promoter by hypermethylation or by inhibition of binding of transcription factors due to point mutations did not contribute to the loss of mRNA and protein in the patient. CONCLUSIONS: Hypermethylation in the Rb promoter region appeared to have little causal effect on HCC.

Detection of hepatitis C virus RNA in circulating immune complexes by RT-PCR.

BACKGROUND/AIMS: We investigate whether hepatitis C virus (HCV) forms a circulating immune complex (CIC) in patients with chronic HCV infection. MATERIALS AND METHODS: We examined HCV-RNA immunoprecipitated with anti-human IgG, A and M antibodies by reverse transcription-polymerase chain reaction. RESULTS: In thirty-nine (91%) of 43 patients, composed of 35 chronic hepatitis (CH) and 8 liver cirrhosis (LC), HCV-RNA was detected in the CIC. All 43 patients analyzed were classified into the following three categories; HCV-RNA was detected only in the supernatant (S pattern, 4 patients), both in the supernatant and the precipitate (SP pattern, 27 patients), and only in the precipitate (P pattern, 12 patients). SP pattern was most common in chronic HCV infection, and the frequency of SP pattern decreased with the progression of liver disease. P pattern was significantly more frequent in patients with higher gamma-globulin levels, histologically indicated LC, and antibody to HCV envelope protein. CONCLUSION: We found that HCV formed a CIC in most patients with chronic HCV infection, and that the formation of CIC might be related to the stage of chronic HCV infection.

Improvement of serum amino acid profile in hepatic failure with the bioartificial liver using multicellular hepatocyte spheroids.

We designed a bioartificial liver support system in which encapsulated multicellular spheroids of rat hepatocytes were utilized as a bioreactor in a hollow fiber cartridge. The spheroids, formed in a positively charged polystyrene dish that contained hormonally defined medium, were encapsulated into microdroplets of agarose that contained about 9 x 10(7) rat hepatocytes. The medium, including 150 mL reservoir volume, was circulated in a closed circuit in which the cartridge was inserted. The pH and levels of dissolved oxygen were monitored and automatically regulated so that they were maintained within a constant range for 72 h. Albumin accumulated in the circuit at the rate of 2.0 mg/L/h in this system. When the bioreactor cells in the system were replaced with Hep G2 cells, a human hepatoblastoma cell line, albumin accumulated at the rate of 0.15 mg/L/h. The spheroids of primary culture hepatocytes had 13 times higher albumin-producing capacity than the aggregates of Hep G2. The serum of a patient with fulminant hepatic failure was circulated in this system with the spheroids of primary culture hepatocytes. The concentration of branched amino acid (BCAA) in the circuit significantly increased during the 48 h circulation, while the concentration of aromatic amino acid (AAA) and methionine decreased. The ratio of BCAA/AAA increased from 0.640 to 0.772, indicating that the hepatocyte spheroids had improved the imbalance of the amino acid profile in the serum. These findings indicate that this system may be a useful model for an artificial liver support. (c) 1996 John Wiley & Sons, Inc.

Immunohistochemical study of proteoglycans in D-galactosamine-induced acute liver injury in rats.

In this study, we carried out an immunohistochemical investigation of time-dependent alterations in the distribution of proteoglycans, and the proliferation profiles of hepatocytes and fat-storing cells (FSCs) in the livers of rats intoxicated with D-galactosamine (GalN). The proliferative cells were analyzed by proliferative cell nuclear antigen (PCNA) staining. In untreated rats, heparan sulfate, dermatan sulfate, and chondroitin/chondroitin sulfate were detected within the portal spaces and the central veins, and, with the exception of chondroitin, also within the reticular fibers. After administration of GalN, the number of PCNA-positive cells (FSCs and hepatocytes) and FSCs increased, reaching maximal on the 2nd and 3rd days, respectively. Heparan sulfate showed complicated changes. Dermatan sulfate decreased in portal spaces from the 2nd to the 3rd day, and in reticular fibers from 12 h to the 6th day. Chondroitin/chondroitin sulfate staining was observed from 2 h to the 6th day in the sinusoidal endothelia, which suggests that the sinusoidal endothelia may produce chondroitin/chondroitin sulfate transiently during liver damage as part of the mechanism of regeneration. Heparan sulfate and chondroitin/chondroitin sulfate were detected in necrotic regions, but dermatan sulfate was not. These observations suggest that heparan sulfate and chondroitin/chondroitin sulfate are involved in cell proliferation or morphogenesis and that the dermatan sulfate plays a role in the differentiation or functional maintenance of cells in liver regeneration.

Structural profile of idiotype, anti-idiotype and anti-anti-idiotype monoclonal antibodies in the HLA-DQ3 antigenic system.

Interest in the characterization of idiotype cascades in the HLA antigenic system has been stimulated by their potential role in the immune response to mismatched HLA allospecificities and in the survival of kidney allografts. Since no information is available about the structural organization of idiotypic cascades in the HLA system, we have sequenced the variable regions of the heavy (VH) and light (VL) chains of mouse anti-HLA-DQ3 monoclonal antibody (mAb) KS13 elicited by cell membrane-bound antigens, of syngeneic anti-HLA-DQ3 mAb S2B154 elicited by anti-idiotypic (anti-id) mAb K03-34 and of five syngeneic anti-id mAb elicited by mAb KS13. mAb KS13 and S2B154, which have been previously shown to be very similar in their specificity and idiotypic profile, share several structural characteristics. Their VH and VL regions are encoded by the same VH, VK and JH genes, display relatively similar V(D)J rearrangements and differ only through a few amino acid substitutions. Among the five anti-id mAb elicited by mAb KS13, mAb R1-38 and R18-9 utilize multiple genetic elements that are different from those used by anti-id mAb KO3-34, K03-256 and K03-335. These results indicate that diverse V region combinations can confer an anti-id specificity in the antigenic system analyzed. mAb K03-34, K03-256 and K03-335 originate from the same B cell clone, since they use the same V, D and J genes and possess identical V(D)J rearrangements. The latter three anti-id mAb differ only by point mutations, which have dramatic effects on the HLA-DQ3 antigen mimicry properties of the three anti-id mAb. mAb K03-34 is the only one to induce anti-HLA-DQ3 antibodies both in syngeneic and xenogeneic hosts. The antigen mimicry properties of anti-id mAb K03-34 depend upon its three-dimensional conformation, since no significant amino acid sequence homology has been found between its VH and VL regions and alpha 1 and beta 1 domains of HLA-DQ3 antigens.

Detection by western blotting of an antibody to the hepatitis C virus E1 envelope protein in sera of patients with chronic liver disease.

We detected an antibody to HCV envelope protein (E1) in sera of patients with HCV-related chronic liver diseases (20 patients with chronic hepatitis and 5 patients with liver cirrhosis) by Western blotting using the fusion protein of E1 envelope protein and beta-galactosidase as an antigen. The antibody to HCV E1 (anti-HCV E1) was detected in 8 (42%) of 19 patients positive for HCV-RNA (16 were positive and 3 were negative for antibody to C100-3) and in 1 (17%) of 6 patients negative for HCV-RNA but positive for antibody to C100-3. HCV-RNA was detected in 8 (89%) of 9 anti-HCV E1 positive sera. The value of alanine aminotransferase was significantly higher in patients positive for anti-HCV E1 than in patients negative for the antibody. Although an antibody to the envelope protein of HCV is suspected to be one of the candidates of virus-neutralizing antibodies, our results suggest this hypothesis appears to be unlikely.

Combination of epidermal growth factor and insulin is required for multicellular spheroid formation of rat hepatocytes in primary culture.

We showed that the combination of epidermal growth factor (EGF) and insulin is an essential supplement to Williams' #E medium for the formation of floating multicellular spheroids in primary culture of rat hepatocytes. Isolated hepatocytes assembled to form floating multicellular spheroids within 96 h through transient assembly of monolayer islands within the initial 24 h in dishes coated with liver-derived proteoglycans. However, the assembly of multicellular spheroids was severely suppressed in the absence of either EGF or insulin. The reduction of spheroid assembly was correlated with decreased attachment and subsequent decreased formation of monolayer islands within 24 h. The minimum amounts of EGF and insulin required for the formation of floating spheroids were 1 ng/ml and 0.4 microgram/ml, respectively. These results suggest that the enhancement of hepatocyte attachment provided by the combination of EGF and insulin during the early phase of culture is required for the formation of floating spheroids.

Fine specificity and idiotype diversity of a murine anti-HLA-DQw3 monoclonal antibody elicited with a syngeneic antiidiotypic monoclonal antibody.

A total of 469 hybridomas was generated from a BALB/c mouse immunized with the syngeneic antiidiotypic mAb KO3-34 that recognizes an idiotope within the Ag-combining site of the immunizing syngeneic anti HLA-DQw3 mAb KS13. One of the hybridomas, named S2B154, was shown with a number of serologic and immunochemical assays to mimic the specificity of anti HLA-DQw3 mAb KS13. This result was unexpected, because the antiidiotypic mAb had been classified as gamma because of the lack of detection of anti-HLA-DQw3 antibodies in sera from BALB/c mice immunized with the antiidiotypic mAb KO3-34. The antiidiotypic mAb S2B154, an IgG1, displays a lower affinity constant to HLA-DQw3 Ag-bearing lymphoid cells than mAb KS13, an IgG2b, but a higher one to antiidiotypic mAb KO3-34. Testing with a panel of antiidiotypic mAb elicited with the mAb KS13 showed that both antiidiotypic mAb S2B154 and mAb KS13 express in their Ag-combining site the idiotopes recognized by the antiidiotypic mAb KO3-256, KO3-335, and R1-38. However, the antiidiotypic mAb S2B154 does not react with the mAb R18-9 that recognizes an idiotope outside the Ag-combining site of mAb KS13. The results we have presented question the validity of the classification of antiidiotypic antibodies based on their ability to inhibit the binding of the corresponding antibodies to Ag and on the specificity of antisera elicited with antiidiotypic antibodies. Furthermore, the hybridomas we have developed in this Id cascade provide us the opportunity to analyze the structural basis of idiotypic Ag mimicry and to evaluate the regulatory properties of antiidiotypic antibodies in the immune response to HLA class II Ag.

Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments.

Adult rat hepatocytes formed floating multicellular spheroids in primary culture in an uncoated plastic dish with a positively charged surface. Cells in the spheroids formed in such a simple way were similar to those formed in dishes coated with proteoglycan fraction isolated from rat liver reticulin fibers; in both cases, cells maintained high ability to produce albumin and poor ability to proliferate in response to epidermal growth factor. Coating dishes with albumin was also helpful in spheroid formation; coating with 2-hydroxymethyl methacrylate resulted in formation of incomplete spheroids. Elimination of serum factors was essential for the formation of spheroids; when cells were washed with serum-containing medium before seeding or if the medium was replaced with a serum-containing medium, spheroid formation was completely inhibited. Collagens, fibronectin, and laminin, all of which promote the adhesion and spreading of hepatocytes on substrates, inhibited spheroid formation. Furthermore, collagens disintegrated spheroids, and cells in the monolayer initiated proliferation. Thus, two distinct, mutually exclusive features of primary culture of adult hepatocytes apparently exist; monolayer culture with proliferative activity in an adherent environment and spheroid culture with poor proliferative activity and high albumin-producing ability in a nonadherent environment.