A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells

Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.     

Drug Resistance of Staphylococci

Induction of chloramphenicol (CM) resistance in Staphylococcus aureus was investigated using 166 CM derivatives and analogues. It was found that 18 compounds of the samples used were able to induce resistance to CM in Staphylococcus aureus S1477 harboring an inducible CM-resistance determinant. Most of the samples which had a nitrophenyl moiety and a D- or DL-threo isomer in its steric configuration were found to have inducer activity for CM resistance. Competent inducers are D-isomer CM derivatives which have another substituent in place of the hydroxyl group at carbon atom 3 in the propanediol of the drug.     

Constitutive release of IL6 by human papillomavirus type 16 (HPV16)-harboring keratinocytes: a mechanism augmenting the NK-cell-mediated lysis of HPV-bearing neoplastic cells.

In the present study we demonstrate that the cultured human keratinocyte cell line (SK-v cells) harboring and expressing integrated human papillomavirus type 16 (HPV16) DNA sequences constitutively releases IL6, which is known as a pleiotropic immunoregulatory cytokine of potential antitumor properties. The presence of IL6 activity in SK-v cell-conditioned media (SK-v CM) was demonstrated by tritiated thymidine incorporation into IL6-dependent B9 murine plasmacytoma cells. The effect on B9 cells was specific since it could be inhibited by anti-IL6 neutralizing antibodies but not by a normal control serum. IL6 did not affect SK-v cell growth; however, it significantly augmented NK cell activity of human peripheral blood lymphocytes against both K562 erytholeukemic and SK-v cells as assessed by 51Cr release assay. SK-v CM displayed NK cell-augmenting activity that copurified with IL6 activity in both size exclusion and anion-exchange HPLC. Furthermore, SK-v cell-derived NK cell stimulatory activity could be neutralized with anti-IL6 antibodies. These results suggest that HPV-harboring neoplastic cells can release IL6 which may indirectly mediate tumor death by augmentation of NK cell activity.     

Biodegradation of leuco derivatives of triphenylmethane dyes by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. CM9

A leuco derivatives of triphenylmethane dyes degrading bacterium, strain CM9, was isolated from an aquafarm field. Based on morphology, physiologic tests, 16S rDNA sequence, and phylogenetic characteristics, it was identified as Sphingomonas sp. This strain was capable of degrading leucomalachite green (LMG), leucocrystal violet and leucobasic fuchsin completely. The relationship between bacterium growth and LMG degradation suggested that strain CM9 could use LMG as the sole source of carbon. The most LMG degradation activity of CM9 crude extract was observed at pH 7.0 and at 30°C. Many metal ions had little inhibition effect on the degradation activity of the crude extract. CM9 also showed strong decolorization of triphenylmethane dyes to their leuco derivatives. GC/MS analysis detected two novel metabolic products, methylbenzene and 4-aminophenol, during the LMG degradation by CM9.     

Mutagenic activities of ten imidazole derivatives in <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

Ten imidazole derivatives were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 both in the absence and presence of metabolic activation by the microsomal fraction S9 mix. In a general manner, derivatives tested exhibited a greater mutagenic activity in the TA100 strain comparing to the responses in TA 98. In the standard plate incorporation assay, 8 of these substances (80%) were found to be mutagenic for at least one of the two strains in the presence or absence of metabolic activation. Two compounds showed positive results in TA98 and 6 compounds were also mutagenic in TA100 without S9. In the presence of S9 mix, all of the 10 substances were non-mutagenic in TA98, whereas 4 compounds were positive in TA100. The results suggested the mutagenic potentials of the imidazole derivatives particularly inducing the reversion of base-pair substitutions. According to the structure-activity relationships phenyl groups in position 2 with different substituents can confer the mutagenic activity of the tested compounds. Methyl groups in different positions of these phenyl substituents can cause different types of mutations. This mutagenic effect is observed more clearly when the phenyl group is inhibited with a nitro group.     

Uncovering Biological Application of Brazilian Green Propolis: A Phenotypic Screening against Schistosoma mansoni

The chemotherapy of schistosomiasis remains centered in the use of praziquantel, however, there has been growing resistant parasites to this drug. Thus, the aim of this work was to evaluate in vitro schistosomicidal activity of the hexanes/dichloromethane 1 : 1 extract of Brazilian green propolis (Pex), as well as its major isolated compounds artepillin C, caffeic acid, coumaric acid and drupanin against Schistosoma mansoni. The Pex was active by displaying an IC₅₀ value of 36.60 (26.26–51.13) μg mL^?1 at 72 h against adult worms of S. mansoni. The major isolated compounds were inactive with IC₅₀ values >100 μM, however, the combination of the isolated compounds (CM) in the same range found in the extract was active with an IC₅₀ value of 41.17 (39.89–42.46) μg mL^?1 at 72 h. Pex and CM induced alteration in the tegument of S. mansoni, and caffeic acid caused alteration in egg's maturation. Pex displayed in vitro activity against adult worms’ and eggs’ viability of S. mansoni, which opens new perspectives to better understand the synergistic and/or additive effects promoted by both Pex extract and CM against schistosomiasis features.     

Chlorella vulgaris (Chlorellaceae) does not secrete autoinhibitors at high cell densities

Filtrates (conditioned medium) from high-density Chlorella vulgaris cultures in photobioreactors were obtained and tested for autoinhibitory activity under different conditions. Exponentially growing cells were inoculated at low initial cell concentration (2 × 10⁵ cells/ml) in 90% conditioned medium (CM) supplemented with 10% fresh medium (FM) at low (atmospheric) CO₂ levels. The time sequence of DNA histograms of cells in CM cultures showed that there is an accumulation of cells with two and four DNA equivalents in the culture over a period of time, signifying a blockage of cells at the division stage of the cell cycle. Examination of the chemical composition of CM showed the presence of high concentrations (> 10 mM) of bicarbonate. Adding similar bicarbonate concentrations to FM were found to have similar effects as CM cultures, causing blockage of cell division, though the intensity of the blocking effect was lower. The bicarbonate-free CM did not show any cell cycle modulating or inhibitory activity. The growth of cells cultivated at high (5%) CO₂ levels in 90% CM supplemented with 10% FM was comparable to 10% FM cultures, indicating nutrient limitation in 90% CM culture. When the 90% CM culture was supplemented with 100% nutrients, the growth rate and final cell concentration was similar to 100% FM culture. Based on these results we conclude that C. vulgaris does not secrete any autoinhibitor(s) or cell cycle modulating compound(s) under the conditions from which the CM was obtained.     

Identification of Peptidases in Highly Pathogenic vs. Weakly Pathogenic Naegleria fowleri Amebae

Naegleria fowleri, a free‐living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse‐passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba‐CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.     

Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells

Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.     

An Investigation of Molecular Targeting of MMP-9 for Endometriosis Using Algal Bioactive Molecules

Endometriosis is a chronic inflammatory disease that occurs due to the presence of endometrial tissue outside the uterine cavity. It affects from 5% to 10% of women of reproductive age. High levels of matrix metalloproteinase (especially MMP-9) have been observed in women suffering from endometriosis. Thus, the aim of this study was to investigate the naturally anti-inflammatory compounds available from an algal source that can target the MMP-9 by various in silico approaches. The target 1L6J (Crystal structure of human matrix metalloproteinase MMP-9) structure was retrieved from the PDB database. Five compounds such as Eckol, Sargafuran, Vitamin E, Docosahexaenoic acid, Fucoidan and Elagolix were selected based on ‘Lipinski’s rule of five’ using the PubChem database. The pharmacokinetics, ADMET properties and biological activity of these compounds were predicted computationally using databases such as PreADME, SWISS-ADME, pkCSM and PASS. Comparative analysis of the bioactive compounds with the target was performed by AutoDock 4.2.6. Using LigPlot v.2.2, the target residues interacting with the compounds were visualised in a 2D manner. Based on the results, Eckol exhibited the highest binding energy value of −7.82 kcal/mol, whereas the Elagolix (control drug) showed a binding energy of −4.88 kcal. We conclude that Eckol can be a potent inhibitor of target MMP-9 with least side effects when compared to the control drug. Hence, this compound can be effectively explored by further in vitro and in vivo studies to develop more effective treatments for Endometriosis.     

Design,synthesis, in vitro antiproliferative activity and apoptosis-inducing studies of 1-(3′,4′,5′-trimethoxyphenyl)-3-(2′-alkoxycarbonylindolyl)-2-propen-1-one derivatives obtained by a molecular hybridisation approach

Inhibition of microtubule function using tubulin targeting agents has received growing attention in the last several decades. The indole scaffold has been recognized as an important scaffold in the design of novel compounds acting as antimitotic agents. Indole-based chalcones, in which one of the aryl rings was replaced by an indole, have been explored in the last few years for their anticancer potential in different cancer cell lines. Eighteen novel (3′,4′,5′-trimethoxyphenyl)-indolyl-propenone derivatives with general structure 9 were synthesized and evaluated for their antiproliferative activity against a panel of four different human cancer cell lines. The highest IC₅₀ values were obtained against the human promyelocytic leukemia HL-60 cell line. This series of chalcone derivatives was characterized by the presence of a 2-alkoxycarbonyl indole ring as the second aryl system attached at the carbonyl of the 3-position of the 1-(3′,4′,5′-trimethoxyphenyl)-2-propen-1-one framework. The structure–activity relationship (SAR) of the indole-based chalcone derivatives was investigated by varying the position of the methoxy group, by the introduction of different substituents (hydrogen, methyl, ethyl or benzyl) at the N-1 position and by the activity differences between methoxycarbonyl and ethoxycarbonyl moieties at the 2-position of the indole nucleus. The antiproliferative activity data of the novel synthesized compounds revealed that generally N-substituted indole analogues exhibited considerably reduced potency as compared with their parent N-unsubstituted counterparts, demonstrating that the presence of a hydrogen on the indole nitrogen plays a decisive role in increasing antiproliferative activity. The results also revealed that the position of the methoxy group on the indole ring is a critical determinant of biological activity. Among the synthesized derivatives, compound 9e, containing the 2-methoxycarbonyl-6-methoxy-N-1H-indole moiety exhibited the highest antiproliferative activity, with IC₅₀ values of 0.37, 0.16 and 0.17?μM against HeLa, HT29 and MCF-7 cancer cell lines, respectively, and with considerably lower activity against HL-60 cells (IC₅₀: 18?μM). This derivative also displayed cytotoxic properties (IC₅₀ values ～1?μM) in the human myeloid leukemia U-937 cell line overexpressing human Bcl-2 (U-937/Bcl-2) via cell cycle progression arrest at the G₂-M phase and induction of apoptosis. The results obtained also demonstrated that the antiproliferative activity of this molecule is related to inhibition of tubulin polymerisation. The presence of a methoxy group at the C5- or C6-position of the indole nucleus, as well as the absence of substituents at the N-1-indole position, contributed to the optimal activity of the indole-propenone-3′,4′,5′-trimethoxyphenyl scaffold.     

Differential induction of chorismate mutase isoforms by elicitors in oat leaves

Avenanthramides, a series of substituted cinnamic acid amides with anthranilate, are phytoalexins in oats (Avena sativa L.). The precursors of avenanthramides, cinnamate and anthranilate, are biosynthesized via the shikimate pathway that branches at chorismate. Chorismate mutase (CM, EC 5.4.99.5) is the first enzyme on the branch that provides the cinnamate part of avenanthramides. The induction of CM was investigated in primary oat leaves using oligo-N-acetylchitooligosaccharides as elicitors. The CM activity started to increase 6 h after elicitation, and reached a maximum by 9 h, being around twice as large as that in control leaves. Among the oligo-N-acetylchitooligosaccharides tested, tetra-, penta-, and hexasaccharides effectively induced the CM activity in a dose-dependent manner. The activity was separated into two major peaks on anion exchange chromatography with Mono Q, indicating that at least two CM isoforms are present in oat leaves. A comparison of elution profiles of CM activity in intact and elicitor-treated leaves revealed that only one CM isoform is responsive to the elicitor. Two CM isoforms in oat leaves were partially purified and characterized. Both CM isoforms were insensitive to l-phenylalanine, l-tyrosine, l-tryptophan, and caffeate. The fractionation of oat cells indicated that both CM isoforms localized in plastids.     

Site-directed mutagenesis of aromatic residues in the carbohydrate-binding module of <Emphasis Type="Italic">Bacillus</Emphasis> endoglucanase EGA decreases enzyme thermostability

The endoglucanase, EGA, from Bacillus sp. AC-1 comprises a glycosyl hydrolase family-9 catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). Seven aromatic residues were subjected to site-directed mutagenesis in both CBM3 and EGA to investigate their roles in enzyme thermostability. The complexes generated by mixing CBMY527G, CBMW532A, or CBMF592G with CM9 each lost their activities after 15 min at 45°C, while the wild-type complex retained >70% activity after 2 h. The mutants EGAY527G, EGAW532A, and EGAF592G showed little activity after 15 min at 60°C, whereas EGA remained 70% active after 2 h. Thus the residues Tyr₅₂₇, Trp₅₃₂, and Phe₅₉₂ contribute not only to CBM3-mediated stability of CM9 but also to EGA thermostability suggesting that hydrophobic interaction between the two modules, independent of covalent linkages, is important for enzyme thermostability.     

Murine osteoclastogenesis suppression using conditioned media produced by melanoma or activated and non‐activated Jurkat‐E6 cells

Conditioned medium (CM) (cell secretome) is a cocktail of growth factors, cytokines, and other soluble mediators secreted by cells into a culture medium. These growth factors are fundamental in many cellular processes such as cell growth, differentiation, and others and the composition of these factors is individual for each cell type. Osteoclasts are large multinucleated cells that are responsible for bone resorption. Immune and cancer cells are known to produce different growth factors, which are able to induce or inhibit osteoclast differentiation. Herein, we evaluated the effect of CM obtained from the supernatant of activated and non‐activated Jukart‐E6 cells, as well as from one murine (B16‐F10) and one human melanoma cell line (SK­MEL­28). To induce osteoclast differentiation, murine bone marrow mononuclear cells were cultured in the presence and absence of differentiation factors (DF), such as macrophage colony‐stimulating factor, prostaglandin E2, receptor activator of nuclear factor‐κB ligand, and CM. We measured the concentration of interleukin 6, tumor necrosis factor‐α and interferon γ (IFN‐γ) in CM that can inhibit or induce osteoclastogenesis. Our study demonstrated that CM obtained from each cell line suppresses or inhibits osteoclasts formation at early and intermediate stages of differentiation in the absence or presence of DF. CM obtained from activated Jurkat‐E6 cells demonstrates a stronger effect when compared with CM from naïve Jurkat‐E6 cells or human and murine melanoma cells. Moreover, CM obtained from activated Jurkat‐E6 cells shows higher secretion of IFN‐γ, which is an inhibitor of osteoclastogenesis, in comparison with CM obtained from the three other cell lines. On the other hand, CM derived from B16‐F10 cells showed a smaller inhibitory effect when compared with CM derived from the other cells.     

Thrombocytopenia in an animal model of malaria is associated with an increased caspase-mediated death of thrombocytes

Infection of mice with Plasmodium Berghei Anka (PbA) leads to a thrombocytopenia, due to a reduced platelet life span, eventually associated with a syndrome of severe or cerebral malaria (CM). Thrombocytopenia was associated with an increase in the number of microparticles (mcp) in plasma. More than >60% of these mcp were of platelet origin, as seen by staining with an anti-platelet antibody. The thrombocytopenia and the amount of mcp were decreased in mice treated with anti CD40L mAb, suggesting that CD40L is the main effector of the thrombocytopenia. Caspase-1, -3, -6, -8, -9 were activated in platelets from infected mice, as seen by the binding of labeled probes or the amount of pro-caspase-3. Treatment of infected mice with the caspases inhibitor ZVAD-fmk decreased the number of mcp and the thrombocytopenia, shoving that platelet caspases are responsible for platelet fragmentation. In addition, the caspase inhibitor also caused a decrease in the mortality associated with CM, indicating a critical role of caspases in the expression of CM.     

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons : II. Developmental aspects

Dissociated sympathetic neurons from the neonatal rat, grown in cell culture in the virtual absence of other cell types, can develop many of the properties expected of differentiated adrenergic neurons including the ability to synthesize and accumulate catecholamines (CA)². However, in the presence of high concentrations of appropriately conditioned medium (CM), the cultures develop the ability to synthesize and accumulate acetylcholine (ACh); correspondingly, their ability to synthesize CA decreases. In this paper several developmental aspects of the CM effect are described. The time course of development of cultures grown with or without CM was followed using synthesis and accumulation of [³H]CA from [³H]tyrosine and production of [³H]ACh from [³H]choline as assays for adrenergic and cholinergic differentiation. The ability to produce CA or ACh developed along parallel time courses in the two sets of cultures, rising primarily during the second week in vitro and reaching a plateau during the fourth week. When CM was used as a cholinergic developmental signal, the sympathetic neurons showed a decreasing response to addition of CM as they matured adrenergically; addition of CM during the third or fourth 10 days in vitro was not as effective in inducing ACh production as addition during the first or second 10 days. Similarly, removal of CM at various times from cultures previously grown in CM showed that the cholinergic induction caused by CM was not easily reversible in older cultures. Thus, as with the adrenergic decision, the cholinergic decision becomes less reversible as the phenotype becomes fully expressed.     

Cold-storage of mixed inoculum of Glomus intraradices enhances root colonization,phosphorus status and growth of hot pepper

Growth response of hot pepper (Capsicum annuum L.) inoculated with the arbuscular mycorrhizal (AM) fungus, Glomus intraradices Schenck and Smith was evaluated in a greenhouse study. Three treatments in a soil-based medium amended with rock phosphate were: (1) control (CON), (2) inoculation of G. intraradices as a freshly prepared soil mixture of spores, hyphae and colonized roots of Sorghum vulgare (FM), and (3) inoculation of the fungus as cold-stored mixed inoculum (CM). Colonization at 14 weeks after inoculation with CM was 42.5%, but was significantly lower with FM (14.5%). Inoculation with G. intraradices as FM and CM increased growth of pepper, and total phosphorus and nitrogen uptake in shoots and roots compared with the CON treatment. Inoculation with CM resulted in significant increases in plant dry weight and chlorophyll concentration compared to the FM and CON treatments. Acid phosphatase activity in the rhizosphere was generally increased by AM fungal treatments. Highest acid phosphatase activity occurred at 14 weeks after inoculation with CM. Alkaline phosphatase activity in the CM treatment was significantly higher compared to that in CON and FM treatments throughout the growth period. Thus, cold storage of mixed inoculum enhanced colonization and growth-promoting activity of G. intraradices compared to freshly prepared inoculum.     

Development of an <Emphasis Type="Italic">in vitro</Emphasis> assay for the investigation of metabolism-induced drug hepatotoxicity

In a number of adverse drug reactions leading to hepatotoxicity drug metabolism is thought to be involved by generation of reactive metabolites from nontoxic drugs. In this study, an in vitro assay was developed for measurement of the impact of metabolic activation of compound on the cytotoxicity toward a human hepatic cell line. HepG2 cells were treated for 6 h with compound in the presence or absence of rat liver S9-mix, and the viability was measured using the MTT test. The cytotoxicity of cyclophosphamide was substantially increased by S9-mix in the presence of NADPH. Three NADPH sources were tested: NADPH (1 mmol/L) or NADPH regenerating system with either NADP⁺/glucose 6-phosphate (G6P) or NADP⁺/isocitrate. All three NADPH sources increased the cytotoxicity of cyclophosphamide to a similar extent. Eight test compounds known to cause hepatotoxicity were tested. For these, only the cytotoxicity of diclofenac was increased by S9 enzymes when an NADPH regenerating system was used. The increased toxicity was NADPH dependent. Reactive drug metabolites of diclofenac, formed by NADPH-dependent metabolism, were identified by LC-MS. Furthermore, an increase in toxicity, not related to enzymatic activity but to G6P, was observed for diclofenac and minocycline. Tacrine and amodiaquine displayed decreased toxicity with S9-mix, and carbamazepine, phenytoin, bromfenac and troglitazone were nontoxic at all tested concentrations, with or without S9-mix. The results show that this method, with measurement of the cytotoxicity of a compound in the presence of an extracellular metabolizing system, may be useful in the study of cytotoxicity of drug metabolites.     

Synthesis of barbituric acid derivatives and their effect on survival of functional hepatocytes from adult rats in primary culture

Summary Ten barbituric acid (BA) derivatives were synthesized and tested for their potency for supporting survival of functional hepatocytes from adult rats in primary culture. Of the 10 BA derivatives, 7 compounds (C-2, 3, 4, 5, 6, 9, and 10) efficiently supported hepatocyte survival for at least 2 wks in primary culture. Especially C-5, 6, and 9 showed excellent efficiency for such action. The optimum concentrations of the BA derivatives for observing the morphological and biochemical effects differed from each other. The maintenance of hepatocytes was attained only in the continuous presence of the BA derivatives in the medium. The morphologic features of hepatocytes surviving in the presence of the BA derivatives resembled those of hepatocytes 24 h after inoculation. The surviving hepatocytes secreted remarkably large amounts of albumin into the culture media. Tyrosine aminotransferase (TAT) activity was higher in the 1-wk-old cultures treated with C-5, 6, and 9 than in the freshly isolated hepatocytes. The addition of dexamethasone (10 μM) caused a 1.7 to 2.1-fold induction in TAT activity. The basal levels of TAT activity and the induction rates increased in the cultures treated with C-5 and 6 from Week 1 to 2 of primary culture.