Chemical Composition and Bioactivity of Dill Seed (Anethum graveolens L.) Essential Oil from Plants Grown under Shading.

This study determined the content and composition of dill seed (Anethum graveolens L.) essential oil under varying light conditions: non-shaded plants in open fields and plants covered with pearl shade nets (40% shade index). Essential oil was extracted using Clevenger hydrodistillation. The essential oil content was 4.63% for non-shaded plants and 4.81% for shaded plants. GC/MS analysis revealed twenty-one and twenty-two components in dill seed from non-shaded and shaded plants, respectively. The terpenic fraction of essential oil from non-shaded plants consisted mainly of oxygen-containing monoterpene derivatives (53.6%), with carvone (46.1%) as the primary component, followed by monoterpene hydrocarbons (46.4%), predominantly limonene (43.8%). Essential oil from shaded plants contained a higher content of carvone (49.8%) and a lower content of limonene (37.8%) compared to essential oil from non-shaded plants. Non-shaded plant essential oil exhibited stronger antioxidant activity (EC50 value: 26.04 mg mL-1) than shaded plant essential oil (54.23 mg mL-1). Dill seed essential oil showed the most potent antimicrobial activity (disc diffusion method) against Escherichia coli (inhibition zone: 15-18 mm). Shaded plants demonstrated a positive influence of essential oil against Klebsiella pneumoniae. Carvone and its derivatives, as the main components, hold significant potential in the food industry and alternative medicines. A practical implication of this study could be higher plant densities or intercropping of dill, as it thrives with minimal light.

Modification of light intensity influence essential oils content, composition and antioxidant activity of thyme, marjoram and oregano.

Thymus vulgaris L. (thyme), Origanum majorana L. (marjoram), and Origanum vulgare L. (oregano) were used to determine whether light modification (plants grown under nets with 40% shaded index or in un-shaded open field) could improve the quantity and quality of essential oils (EOs) and antioxidant activity. The yield of EOs of thyme, marjoram, and oregano obtained after 120 min of hydrodistillation was 2.32, 1.51, and 0.27 mL/100 g of plant material, respectively. At the same time under shading conditions plants synthetized more EOs (2.57, 1.68, and 0.32 mL/100 g of plant material). GC/MS and GC/FID analyses were applied for essential oils determinations. The main components of the thyme essential oil are thymol (8.05-9.35%); γ-terpinene (3.49-4.04%); p-cymene (2.80-3.60%) and caryophyllene oxide (1.54-2.15%). Marjoram main components were terpinene 4-ol (7.44-7.63%), γ-terpinene (2.82-2.86%) and linalool (2.04-2.65%) while oregano essential oil consisted of the following components: caryophyllene oxide (3.1-1.93%); germacrene D (1.17-2.0%) and (E)-caryophyllene (1.48-1.1%). The essential oil from thyme grown under shading (EC50 value after 20 min of incubation) have shown the highest antioxidant activity - 0.85 mg mL-1 in comparison to marjoram and oregano (shaded plants EC50 19.97 mg mL-1 and 7.02 mg mL-1 and unshaded, control plants EC50 54.01 mg mL-1 and 7.45 mg mL-1, respectively). The medicinal plants are a good source of natural antioxidants with potential application in the food and pharmaceutical industries. For production practice, it can be recommended to grow medicinal plants in shading conditions to achieve optimal quality parameters.

The Correlation between Extracellular Heat Shock Protein 70 and Lipid Metabolism in a Ruminant Model.

Metabolic stress in early lactation cows is characterized by lipolysis, ketogenesis, insulin resistance and inflammation because of negative energy balance and increased use of lipids for energy needs. In this study the relationship between lipid metabolite, lipid-based insulin resistance, and hepatocyte functionality indexes and tumor necrosis factor alpha (TNF-α) with extracellular heat shock protein 70 (eHsp70) was investigated. The experiment included 50 cows and all parameters were measured in blood serum. In cows with a more pronounced negative energy balance, the following was determined: a higher concentration of eHsp70, TNF-α, non-esterified fatty acid (NEFA), beta-hydroxybutyrate (BHB), NEFA to insulin and NEFA to cholesterol ratio and lower concentration of cholesterol, very low-density lipoproteins (VLDL), low density lipoproteins (LDL) and liver functionality index (LFI). The eHsp70 correlated negatively with the values of cholesterol, VLDL, LDL, and triglycerides, while correlated positively with the level of NEFA and BHB. A higher concentration of eHsp70 suggests the development of fatty liver (due to a higher NEFA to cholesterol ratio and lower LFI) and insulin resistance (due to a lower revised quantitative insulin sensitivity check index RQUICKI-BHB and higher NEFA to insulin ratio). The eHsp70 correlated positively with TNF-α. Both TNF-α and eHsp70 correlated similarly to lipid metabolites. In cows with high eHsp70 and TNF-α values we found higher concentrations of NEFA, BHB, NEFA to insulin and NEFA to cholesterol ratio and a lower concentration of triglycerides and VLDL cholesterol compared to cows that had only high TNF-α values. Based on the positive correlation between eHsp70 and TNF-α, their similar relations, and the additional effect of eHsp70 (high TNF-α + eHsp70 values) on lipid metabolites we conclude that eHsp70 has pro-inflammatory effects implicating lipolysis, fatty liver, and fat tissue insulin resistance.

Lower concentrations of curcumin inhibit Her2-Akt pathway components in human breast cancer cells, and other dietary botanicals potentiate this and lapatinib inhibition.

Her2-dependent breast cancer is treated with pharmacological drugs (eg, Herceptin, lapatinib) that target Her2 signaling. Curcumin has emerged as a potential co-treatment for this and other cancers, but prior studies have focused on non-attainable concentrations. Here we test the hypothesis that attainable in vivo levels of dietary curcumin can reduce Her2 signaling. Consistent with previous studies, higher dose curcumin (18 µmol/L) inhibits Her2-Akt pathway signaling (pHer2, total Her2 and pAkt levels) and cell growth using AU565 human breast cancer cells. We then examined lower, more physiologically relevant concentrations of curcumin, alone and in combination with other dietary botanicals (quercetin and OptiBerry fruit extract). At 4 µmol/L, curcumin reduced Her2 signaling, and even more when combined with quercetin or OptiBerry. At 1.5 µmol/L curcumin, pHer2 and Her2 (but not pAkt) were reduced, with all three pathway markers reduced more in the presence of quercetin. We also found that 1.5 µmol/L curcumin strongly potentiated lapatinib inhibition of Her2-Akt pathway signaling, and more so for pAkt, when combined with quercetin plus OptiBerry (CQO). Parallel analyses revealed cell growth inhibition at 18 and 4 µmol/L but not 1.5 µmol/L curcumin, and potentiation of 1.5 µmol/L curcumin growth arrest with other botanicals +/- lapatinib. These studies demonstrate that a physiological attainable level of curcumin (1.5 µmol/L) can reduce some components of the critical Her2-Akt pathway; that even more complete inhibition can be achieved by combination with other dietary botanicals; and that curcumin and other botanicals can potentiate the action of the Her2-cancer metastatic drug lapatinib, in turn suggesting the potential anti-cancer clinical use of these botanicals.

Effect of shading and grafting on yield and quality of tomato.

BACKGROUND: The need to increase marketable tomato yields and decrease losses due to sunburn and disease during the summer motivates farmers to adopt additional cultural practices such as shading and grafting. To investigate complex interactions among grafting, shading, and tomato cultivar, grafted and ungrafted tomatoes (cv. 'Optima' F1 and cv. 'Big Beef' F1 ) were grown in the soil under net-house cover, using pearl and red nets, and in unshaded conditions (open fields). Tomato fruit at the red stage of maturity were used for the analysis of quality traits, and total and marketable yields were recorded during the whole production season. RESULTS: Grafting and shading in tomato production might be considered as cultivation practices to increase the marketable tomato yield. A decrease in sugar content increased the uptake of some micro elements (Fe and Zn) and macro elements (Ca). In some cases, firmer and less elastic skin may be expected due to grafting. Shading with pearl net might result in fruit with lower firmness and higher total, and particularly malic, acid content. CONCLUSION: Shading with colored nets and grafting provide alternative strategies for achieving higher fruit yields and avoiding or reducing a decrease in tomato quality caused by environmental stresses such as excessive radiation and temperature in the summer cropping season. © 2019 Society of Chemical Industry.

Comparison of survivor scores for differentiation therapy of cancer to those for checkpoint inhibition: Half full or half empty.

Differentiation therapy is directed to the self-renewing cancer stem cells, as well as their progeny transit amplifying cells, to force them to mature to terminal differentiation. Differentiation therapy is effective in treatment of neuroblastomas and myeloid leukemias. Checkpoint inhibition therapy removes blocks to cancer reactive T-killer cells and allows them to react to malignant cells and limit the growth of cancer. The percentage of patients with a given cancer that responds to either therapy is less than hoped for, and the duration of response is variable. Multiplying the response rate (percentage of patients responding to therapy) by the duration of response may be used to derive a survival score for patients treated with differentiation therapy or checkpoint inhibition. By this criterion, differentiation therapy gives better survival scores than checkpoint inhibition. Yet, checkpoint inhibition is considered a great success, mostly because it may be applied to many different types of cancer, and differentiation therapy is considered relatively ineffective because it is limited to a few specific cancers. On the other hand, the cost of checkpoint inhibition treatment is 10-20 times more per patient than that of differentiation therapy. Hopefully, future combined treatments and advances in both approaches will increase the effectiveness of these cancer treatments.

Control (Native) and oxidized (DeMP) mitochondrial RNA are proinflammatory regulators in human.

Inflammation is implicated in a wide range of disorders, and thought to be involved in most leading causes of death today in the United States with high associated costs. New insights into better understanding its etiology, detection and prevention are thus of major importance in health care. One emerging field providing such insights has been the identification of DAMPs, or damage-associated molecular patterns. We have studied DAMPs within the context of degraded and oxidized mitochondrial DNA and RNA ("DeMP"), most recently demonstrating potent mitochondrial RNA (mtRNA) immunogenic response in mouse macrophages. Here, we extend these studies to assess the proinflammatory role of mitochondrial control (native) and oxidized RNA using human RNA and cells. THP-1 macrophage mtRNA triggered a proinflammatory response (induction of IL-6 and TNFα) when transfected into the same cells. Modestly oxidized mtRNA (DeMP RNA) but not cytoplasmic RNA induced a similar response, in contrast to attenuated immunogenicity previously observed with more oxidized DeMP RNA. This DeMP RNA may also cause a mild prooxidant stress. The proinflammatory effects of mtRNA was significantly reduced following pretreatment with RNases specific for single and double stranded RNA, implicating these forms of mtRNA in proinflammatory response. The natural nucleic acid-encapsulating peptide LL-37 also triggered a proinflammatory effect in the presence of control mtRNA and DeMP RNA. Finally, human blood plasma RNA exhibits proinflammatory activity. These results provide new insights into the immunostimulation of mitochondrial RNA including its activity in human cells; identify human plasma RNA as proinflammatory; and provide further evidence that oxidized DeMP mtRNA acts as a sensitive and broad-spectrum sensor and regulator of mitochondrial oxidative stress.

Participation of liver stem cells in cholangiocarcinogenesis after aflatoxin B1 exposure of glutathione S-transferase A3 knockout mice.

Aflatoxin B1, arguably the most potent human carcinogen, induces liver cancer in humans, rats, trout, ducks, and so on, but adult mice are totally resistant. This resistance is because of a detoxifying enzyme, mouse glutathione S-transferase A3, which binds to and inactivates aflatoxin B1 epoxide, preventing the epoxide from binding to DNA and causing mutations. Glutathione S-transferase A3 or its analog has not been detected in any of the sensitive species, including humans. The generation of a glutathione S-transferase A3 knockout (represented as KO or -/-) mice has allowed us to study the induction of liver cancer in mice by aflatoxin B1. In contrast to the induction of hepatocellular carcinomas in other species, aflatoxin B1 induces cholangiocarcinomas in GSTA3-/- mice. In other species and in knockout mice, the induction of liver cancer is preceded by extensive proliferation of small oval cells, providing additional evidence that oval cells are bipolar stem cells and may give rise to either hepatocellular carcinoma or cholangiocarcinoma depending on the nature of the hepatocarcinogen and the species of animal. The recent development of mouse oval cell lines in our laboratory from aflatoxin B1-treated GSTA3-/- mice should provide a new venue for study of the properties and potential of putative mouse liver stem cells.

Characterization of liver injury, oval cell proliferation and cholangiocarcinogenesis in glutathione S-transferase A3 knockout mice.

We recently generated glutathione S-transferase (GST) A3 knockout (KO) mice as a novel model to study the risk factors for liver cancer. GSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of aflatoxin B1 (AFB1), confirming the crucial role of GSTA3 in resistance to AFB1. We now report histopathological changes, tumor formation, biochemical changes and gender response following AFB1 treatment as well as the contribution of oxidative stress. Using a protocol of weekly 0.5 mg AFB1/kg administration, we observed extensive oval (liver stem) cell (OC) proliferation within 1-3 weeks followed by microvesicular lipidosis, megahepatocytes, nuclear inclusions, cholangiomas and small nodules. Male and female GSTA3 KO mice treated with 12 and 24 weekly AFB1 injections followed by a rest period of 12 and 6 months, respectively, all had grossly distorted livers with macro- and microscopic cysts, hepatocellular nodules, cholangiomas and cholangiocarcinomas and OC proliferation. We postulate that the prolonged AFB1 treatment leads to inhibition of hepatocyte proliferation, which is compensated by OC proliferation and eventually formation of cholangiocarcinoma (CCA). At low-dose AFB1, male KO mice showed less extensive acute liver injury, OC proliferation and AFB1-DNA adducts than female KO mice. There were no significant compensatory changes in KO mice GST subunits, GST enzymatic activity, epoxide hydrolase, or CYP1A2 and CYP3A11 levels. Finally, there was a modest increase in F2-isoprostane and isofuran in KO mice that confirmed putative GSTA3 hydroperoxidase activity in vivo for the first time.

Survival of irradiated recipient mice after transplantation of bone marrow from young, old and "early aging" mice.

Bone marrow transplantation is used to examine survival, hematopoietic stem cell function and pathology in recipients of young and old wild type bone marrow derived stem cells (BMDSCs) as well as cells from p53-based models of premature aging. There is no difference in the long term survival of recipients of 8 week-old p53+/m donor cells compared to recipients of 8 week-old wild-type (WT) donor cells (70 weeks) or of recipients of 16-18 weeks-old donor cells from either p53+/m or WT mice. There is shorter survival in recipients of older versus younger WT donor bone marrow, but the difference is only significant when comparing 8 and 18 week-old donors. In the p44-based model, short term survival/engraftment is significantly reduced in recipients of 11 month-old p44 donor cells compared to 4 week-old p44 or wild type donor cells of either age; mid-life survival at 40 weeks is also significantly less in recipients of p44 cells. BMDSCs are readily detectable within recipient bone marrow, lymph node, intestinal villi and liver sinusoids, but not in epithelial derived cells. These results indicate that recipients of young BMDSCs may survive longer than recipients of old bone marrow, but the difference is marginal at best.

Effect of coloured shade-nets on plant leaf parameters and tomato fruit quality.

BACKGROUND: The concept of photo-selective netting using commercial cultivation practices was studied in a tomato (Solanum lycopersicum 'Vedetta') summer cultivation in south Serbia (under high solar radiation 910 W m(-2) , with a photosynthetic photon flux density of 1661 µmol m(-2) s(-1) ), under four different coloured shade-nets (pearl, red, blue and black) with 40% relative shading. The aim of the study was to determine how different environmental control technologies (coloured shade-nets as screen house or plastic-house integrated with coloured shade-nets) could influence plant parameters, production and quality traits in tomato fruits cultivated in south Serbia (Balkan region). RESULTS: The leaf area index (LAI) ranged from 4.6 to 5.8 in open field and plastic tunnels plants (control) with maximum LAI values of 7.9-8.2 in net houses with red colour nets. Shade-grown leaves generally have higher total chlorophyll and carotenoids content than do control leaves. Pericarp thickness was significantly higher tomatoes grown under pearl (7.215.82 µm), red (7099.00 µm) and blue nets (6802.29 µm) compared to other treatments and to control (6202.48 µm). The highest concentration of lycopene was detected in tomatoes grown in plastic houses integrated with red colour nets (64.9 µg g(-1) fresh weight). The plastic house and open field (control) tomato production had a taste index mean value of 1.09-1.10. This is significantly higher than the values determined for the treatments with different coloured shade-nets. CONCLUSION: These results show that red and pearl photo-selective nets create optimal growing conditions for the growth of the plant and produce fruits with thicker pericarp, the highest lycopene content, a satisfactory level of taste index and can be further implemented within protected cultivation practices.

Genetic or pharmacologic activation of Nrf2 signaling fails to protect against aflatoxin genotoxicity in hypersensitive GSTA3 knockout mice.

Mice are resistant to aflatoxin hepatotoxicity, primarily due to high expression of glutathione S-transferases (GSTs), and in particular the GSTA3 subunit. Nuclear factor erythroid 2 related factor 2 (Nrf2) signaling, which controls a broad-based cytoprotective response, was activated either genetically or pharmacologically in an attempt to rescue GSTA3 knockout mice from aflatoxin genotoxicity. Genetic activation of Nrf2 signaling was attained in a GSTA3: hepatocyte-specific Keap1 double knockout (DKO) mouse whereas pharmacologic activation of Nrf2 was achieved through pretreatment of mice with the triterpenoid 1-[2-cyano-3-,12-dioxoleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) prior to aflatoxin B1 exposure. Following oral treatment with aflatoxin, urine was collected from mice for 24 h and hepatic and urinary aflatoxin metabolites then quantified using isotope dilution-mass spectrometry. Although Nrf2 was successfully activated genetically and pharmacologically, neither means affected the response of GSTA3 knockout mice to chemical insult with aflatoxin. Hepatic aflatoxin B1-N(7)-guanine levels were elevated 120-fold in GSTA3 knockout mice compared with wild-type and levels were not attenuated by the interventions. This lack of effect was mirrored in the urinary excretion of aflatoxin B1-N(7)-guanine. By contrast, urinary excretion of aflatoxin B1-N-acetylcysteine was >200-fold higher in wild-type mice compared with the single GSTA3 knockout or DKO mouse. The inability to rescue GSTA3 knockout mice from aflatoxin genotoxicity through the Nrf2 transcriptional program indicates that Gsta3 is unilaterally responsible for the detoxication of aflatoxin in mice.

Preparation of epithelial and mesenchymal stem cells from murine mammary gland.

The mammary gland is a complex organ consisting of multiple cell types that undergo extensive remodeling during pregnancy and involution, cyclical changes that suggest the existence of a resident stem cell population that is responsible for remarkable tissue regeneration. The basic functional unit of the mammary gland is the terminal duct lobular unit, which invades the stromal tissue (fat, connective tissue, blood vessels, etc.). Luminal epithelial cells line the ducts while outer myoepithelial cells secrete the basal lamina that separates the mammary gland parenchyma from the mesenchymal cells of the stroma. Within the epithelial cell population of the ducts resides the mammary gland stem cells and it is believed that this population is the origin of the mammary gland cancer stem cells as well. In the mouse, epithelial stem cells can be separated from mesenchymal cells on the basis of CD24, CD44, and CD49f expression. This allows for the determination of both normal and cancer stem cell potential of these two populations and permits investigation into their interaction in tumor development.

Age dependence of oval cell responses and bile duct carcinomas in male fischer 344 rats fed a cyclic choline-deficient, ethionine-supplemented diet.

UNLABELLED: The age dependence of the oval cell response and bile duct carcinomas of male F344 rats exposed to a cyclic choline deficiency-ethionine (CDE) diet (2 weeks on, 1 week off) supports the concept of loss of potential of liver stem cells to form cancers with aging. Livers of rats exposed at 3 weeks of age demonstrated a robust and widespread oval cell proliferation followed by cholangiofibrosis and bile duct metaplasia with extensive mucinous cysts throughout all lobes, and induction of cholangiocarcinomas (CCAs) in seven of eight rats. Livers of rats exposed beginning at 8 weeks of age had much less oval cell response and cholangiofibrosis with only 1 of 15 rats developing a CCA. Livers in old (10-12 months when started) rats remained virtually unaffected, with minimal oval cell proliferation, only occasional and small foci of ductular dysplasia, and none of 16 rats developed CCAs. In contrast to most published studies using uninterrupted choline deficiency plus a carcinogen, hepatocellular carcinoma (HCC) was not observed under the conditions of this study. CONCLUSION: With aging, male F344 rats exposed to cyclic CDE diet display a diminished oval cell response and fewer CCAs. The absence of HCC is possibly due to the fact that during cyclic CDE, the week off may allow putative liver stem cells to avoid death or differentiation and survive to give rise to CCAs, whereas with continuous CDE exposure, the stem cells are forced to differentiate and develop into HCCs with relatively few CCAs.

Direct and indirect contribution of bone marrow-derived cells to cancer.

Stromal-epithelial interactions may control the growth and initiation of cancers. Here, we not only test the hypothesis that bone marrow-derived cells may effect development of cancers arising from other tissue cells by forming tumor stroma but also that sarcomas may arise by transformation of stem cells from the bone marrow and epithelial cancers may arise by transdifferentiation of bone marrow stem cells to epithelial cancers. Lethally irradiated female FVB/N mice were restored with bone marrow (BM) transplants from a male transgenic mouse carrying the polyoma middle T-oncoprotein under the control of the mouse mammary tumor virus promoter (MMTV-PyMT) and followed for development of lesions. All of 8 lethally irradiated female FVB/N recipient mice, restored with BM transplants from a male MMTV-PyMT transgenic mouse, developed Y-chromosome negative (Y-) cancers of various organs surrounded by Y+ stroma. One of the female FVB/N recipient mice also developed fibrosarcoma and 1, a diploid breast adenocarcinoma containing Y chromosomes. In contrast, only 1 of 12 control female mice restored with normal male BM developed a tumor (lymphoma) during the same time period. These results indicate not only that the transgenic BM-derived stromal cells may indirectly contribute to development of tumors in recipient mice but also that sarcomas may arise by transformation of BM stem cells and that breast cancers arise by transdifferentiation of BM stem cells, presumably by mesenchymal-epithelial transition.

Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1.

Aflatoxin B1 (AFB1) is a major risk factor for hepatocellular carcinoma (HCC) in humans. However, mice, a major animal model for the study of AFB1 carcinogenesis, are resistant, due to high constitutive expression, in the mouse liver, of glutathione S-transferase A3 subunit (mGSTA3) that is lacking in humans. Our objective was to establish that a mouse model for AFB1 toxicity could be used to study mechanisms of toxicity that are relevant for human disease, i.e., an mGSTA3 knockout (KO) mouse that responds to toxicants such as AFB1 in a manner similar to humans. Exons 3-6 of the mGSTA3 were replaced with a neomycin cassette by homologous recombination. Southern blotting, RT-PCR, Western blotting, and measurement of AFB1-N(7)-DNA adduct formation were used to evaluate the mGSTA3 KO mice. The KO mice have deletion of exons 3-6 of the mGSTA3 gene, as expected, as well as a lack of mGSTA3 expression at the mRNA and protein levels. Three hours after injection of 5 mg/kg AFB1, mGSTA3 KO mice have more than 100-fold more AFB1-N(7)-DNA adducts in their livers than do similarly treated wild-type (WT) mice. In addition, the mGSTA3 KO mice die of massive hepatic necrosis, at AFB1 doses that have minimal toxic effects in WT mice. We conclude that mGSTA3 KO mice are sensitive to the acute cytotoxic and genotoxic effects of AFB1, confirming the crucial role of GSTA3 subunit in protection of normal mice against AFB1 toxicity. We propose the mGSTA3 KO mouse as a useful model with which to study the interplay of risk factors leading to HCC development in humans, as well as for testing of additional possible functions of mGSTA3.

Hepatocyte nuclear factor-1 as marker of epithelial phenotype reveals marrow-derived hepatocytes, but not duct cells, after liver injury in mice.

The potential bone marrow origin of hepatocytes, cholangiocytes, and ductal progenitor cells in the liver was examined in female mice after transplantation of bone marrow cells from male green fluorescent protein (GFP) transgenic donors. Following stable hematopoietic engraftment, the livers of the recipients were injured with carbon tetrachloride (CCl(4), with or without local irradiation of the liver) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC, with or without local irradiation of the liver). The presence of numerous marrow-derived, GFP-positive inflammatory cells had the potential to lead to erroneous interpretation of marrow-derived hepatocytes, cholangiocytes, and ductal progenitor cells. Identification of marrow-derived ductal progenitor or cholangiocyte phenotype using colocalization of GFP or Y chromosome with pancytokeratin staining also failed to distinguish epithelial cells from closely apposed inflammatory cells. To address this inadequacy, we developed a rigorous new immunofluorescence protocol to identify marrow-derived epithelial cells in the liver using Y chromosome (donor marker) and hepatocyte nuclear factor-1 (HNF1, a nuclear marker of liver epithelial, nonhematopoietic phenotype). Using the Y/HNF1 method, rare (approximately one in 20,000) hepatocytes in female mice transplanted with male bone marrow contained a donor-derived Y chromosome. On the other hand, no Y chromosomes were found in cholangiocytes or ductal progenitor cells in mice with liver injury due to DDC or CCl(4). The use of a nuclear marker of mature hepatocytes or cholangiocytes, such as HNF1, improves discrimination of marrow-derived epithelial cells in tissue sections.

Separate origins of hepatitis B virus surface antigen-negative foci and hepatocellular carcinomas in transgenic HBsAg (alb/psx) mice.

We have examined the development and transgene expression in liver lesions of transgenic mice bearing the hepatitis B surface antigen (HBsAg) gene of hepatitis B virus under the control of the albumin promoter (alb/psx) to study liver regeneration and hepatocellular carcinoma (HCC) associated with hepatitis B virus infection. Storage of the HBsAg in the endoplasmic reticulum precedes loss of liver cells and regenerative hyperplastic nodules that do not express HBsAg. Histological analysis indicated that HBsAg-negative foci and nodules arose from liver progenitor cells in the portal zone and lacked mRNA expression. Genomic DNA from eight of nine HBsAg-negative laser capture-excised liver foci showed loss of part of the alb/psx gene, whereas no loss of the actin gene was observed. The alb/psx DNA was intact in adjacent HBsAg-positive tissue. Sequencing of polymerase chain reaction products suggested that alterations in the HBsAg transgene in HBsAg-negative foci occurred via large-scale deletions as opposed to single-site mutations. Southern blot analysis of HCC from 2-year-old transgenic HBsAg mice, however, revealed an intact alb/psx gene. Thus, HBsAg-negative progenitor cells with deletions in the transgene appear to be responsible for compensatory regeneration of the liver, whereas HCCs arise from clonal expansion of hepatocytes with intact alb/psx transgenes.

Reduced expression of p27kip1 and increased hepatocyte proliferation in p53-deficient mice.

Livers from wild-type and p53-deficient mice were analyzed for the expression of cell-cycle regulatory proteins in an attempt to determine the mechanism for the increased proliferation of liver cells in p53-deficient mice associated with enhanced susceptibility to aflatoxin-induced liver cancer. The most striking difference found was a significant reduction of the cyclin-dependent kinase inhibitor p27(kip1) in the livers of 3-mo-old p53-/- mice, whereas only small changes were found in the expression of cyclins, cyclin-dependent kinases, and the inhibitors p21(cip1) and p16(ink4a). Relative to wild-type liver, the amounts of p27(kip1) mRNA were reduced at both 1 and 3 mo, whereas the levels of p27(kip1) protein were decreased only at 3 mo. These results identify an uncharacterized link between the expression of p53 and p27(kip1) that may involve both transcriptional and post-transcriptional regulation and allow hepatocytes to continue to proliferate after 3 wk of age. We postulate that this increased proliferation leads to increased susceptibility to aflatoxin-induced hepatocarcinogenesis.