Williams Medium Research Articles

Selenoprotein V Protects Against Oxidative Stress, Endoplasmic Reticulum Stress, and Apoptosis

Abstract Objectives Selenoprotein V (SELENOV) contains a thioredoxin-like fold and a conserved CxxU motif with a potential redox function. Three experiments were performed to assess its in vivo and in vitro roles and mechanisms in coping with different oxidant insults. Methods In Expt.1, SELENOV knockout (KO) and wildtype (WT) mice (male, 8-wk old) were given an IP injection of saline, diquat (DQ, 12.5 mg/kg), or acetaminophen (APAP, 300 mg/kg) (n = 10), and killed 5 h after the injection to collect liver and blood. In Expt. 2, primary hepatocytes were isolated from the 2 genotypes, cultured in complete Williams's medium E, and treated with DQ (0, 0.25 and 0.75 mM) and APAP (0, 1, 3, and 6 mM) for 12 h. In Expt. 3, 293 T cells were transfected with a control plasmid (GFP) or the plasmid containing Selenov gene (full length, OE) and treated with APAP (0, 1, 2, and 4 mM) for 24 h or H2O2 (0.1, 0.2, and 0.4 mM) for 12 h. Results In Expt. 1, the DQ and APAP injections caused greater (P < 0.05) rises in serum alanine aminotransferase activities, hepatic malondialdehyde (MDA) and carbonyl contents, endoplasmic reticulum (ER) stress-related proteins (BIP and CHOP), apoptosis-related proteins (FAK and caspase 9), and 3-nitrotyrosine, along with lower total anti-oxidizing-capability (T-AOC) and severer hepatocyte necrosis in the central lobular areas, in the KO than in the WT. In Expt. 2, the DQ and APAP treatments induced elevated (P < 0.05) cell death (20–40%), MDA contents (25–35%), and decreased (P < 0.05) T-AOC (50–65%) in the KO hepatocytes than in the WT cells. The KO hepatocytes treated with APAP displayed a sharp decline (P < 0.05) in cellular total respiration ability than the WT cells. In Expt. 3, the OE cells had greater viability and T-AOC and lower reactive oxygen species, MDA, and carbonyl contents after the APAP and H2O2 exposures (all at P < 0.05) than the controls. Moreover, the OE cells had greater (P < 0.05) redox enzyme activities (GPX, TrxR, and SOD), and lower (P < 0.05) expressions of ER stress-related genes (Atf4, Atf6, Bip, Xpp1t, Xbp1s, and Chop) and proteins (BIP, CHOP, FAK, caspase 9) than the controls after the treatment of H2O2 (0.4 mM). Conclusions Our data revealed the in vivo and in vitro roles and mechanisms of SELENOV in protecting against oxidative stress, ER stress, and apoptosis induced by pro-oxidants. Funding Sources This research is supported in part by an NSFC grant #31,320,103,920.

Assessment of Hair Growth Treatment with the Consciousness Energy Healing Treated Williams Medium E Using Mouse Vibrissae Hair Follicle Organ Culture

Hair is playing an interesting part in human for social and sexual communication. Loss of hair follicle leads to various skin disorders. For this consequence, the present study has investigated the potential of the Biofield Energy Healing (The Trivedi Effect®) Treated test item (William’s Medium E) on the vibrissae hair follicle organ culture cells for the assessment of hair cell growth and development in vitro. The test item was divided into two parts. One part was defined as the untreated test item, where no Biofield Energy Treatment provided, while the other part was defined as the Biofield Energy Treated test item, which received the Biofield Energy Healing Treatment by renowned Biofield Energy Healer, Mahendra Kumar Trivedi. The study parameters like bulb thickness and formation of telogen were assessed using cell-based assay with the help of UTHSCSA Image tool version 3. The experimental results showed that the untreated test item group showed 20.9% and 28.2% increased bulb thickness on day 5 and 7, respectively compared to the day 1, while did not produce telogen follicles upto day 7. Besides, the percentage of telogen follicle was found as 43%, 57%, and 71% on day 3, 5, and 7, respectively of the Biofield Energy Treated test item group compared to the day 1. The overall results demonstrated that the Biofield Energy Treatment has the potential for hair growth promotion as evident via increased the formation of telogen. Therefore, the Biofield Energy Healing (The Trivedi Effect®) Treatment might be useful as a hair growth promoter for various treatment of skin injuries and skin-related disorders like necrotizing fasciitis, actinic keratosis, sebaceous cysts, diaper rash, decubitus ulcer etc.

Conditioned Media Derived from Human Adipose Tissue Mesenchymal Stromal Cells Improves Primary Hepatocyte Maintenance.

Objective Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells (MSCs) to improve in vitro maintenance conditions for hepatocytes. Materials and Methods In this experimental study, following serum deprivation, human adipose tissue-derived MSCs (hAT-MSCs) were cultured for 24 hours under normoxic (N) and hypoxic (H) conditions. Their conditioned media (CM) were subsequently collected and labeled as N-CM (normoxia) and H-CM (hypoxia). Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal (William’s) medium supplemented with 4% N-CM or H-CM. Untreated William’s and hepatocyte-specific media (HepZYM) were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells. Results We observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) assay. Indocyanine green (ICG) uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin (Alb) and urea secretion compared to the other groups (P<0.0001). However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor (VEGF) in the H-CM group compared to the N-CM group (P=0.063). ConclusionThe enrichment of William’s basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture.

Vitamin C acts as a hepatoprotectant in carbofuran treated rat liver slices in vitro

Carbamates, most commonly used pesticides in agricultural practices, have been reported to produce free radicals causing deleterious effects in animals. The present study was designed to assess the carbofuran induced oxidative stress in rat liver slices in vitro and also to evaluate protective role of vitamin C by incubating them in Krebs-Ringer HEPES Buffer (KRHB) containing incubation media (Williams medium E (WME) supplemented with glucose and antibiotics) with different concentrations of carbofuran. The results demonstrated that carbofuran caused significant increase in lipid peroxidation and inhibition in the activity of hepatic superoxide dismutase (SOD) in concentration dependent manner. The data with incubation medium reflected that carbofuran at lowest concentration caused an increase in SOD activity followed by its inhibition at higher concentration. Carbofuran treatment caused inhibition in the activity of catalase in liver slices and WME incubation medium. Pre-incubation of liver slices and the WME media with vitamin C restored the values of biochemical indices tested. The results indicated that carbofuran might induce oxidative stress in hepatocytes. The pretreatment with vitamin C may offer hepatoprotection from toxicity of pesticide at low concentration only.

Equine hepatocytes: isolation, cryopreservation, and applications to invitro drug metabolism studies.

Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Clint) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two‐step collagenase perfusion method, with an average cell yield of 2.47 ± 2.62 × 106 cells/g of perfused liver tissue and viability of 84.1 ± 2.62%. These cells were cryopreserved with William's medium E containing 10% fetal bovine serum with 10% DMSO. The viability of recovered cells, after a 30% Percoll gradient, was 77 ± 11% and estimated recovery rate was approximately 27%. These purified cells were used to determine the in vitro Clint of three drugs used in equine medicine; omeprazole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopreserved suspensions gave a comparable estimation of Clint compared to fresh cells for these three drugs as well as producing the same metabolites. This work paves the way for establishing a bank of cryopreserved equine hepatocytes that can be used for estimating pharmacokinetic parameters such as the hepatic metabolic in vivo clearance of a drug as well as producing horse‐specific drug metabolites.

Machine perfusion enhances hepatocyte isolation yields from ischemic livers

BackgroundHigh-quality human hepatocytes form the basis of drug safety and efficacy tests, cell-based therapies, and bridge-to-transplantation devices. Presently the only supply of cells derives from an inadequate pool of suboptimal disqualified donor livers. Here we evaluated whether machine perfusion could ameliorate ischemic injury that many of these livers experience prior to hepatocyte isolation. MethodsNon-heparinized female Lewis rat livers were exposed to an hour of warm ischemia (34°C) and then perfused for 3h. Five different perfusion conditions that utilized the cell isolation apparatus were investigated, namely: (1) modified Williams Medium E and (2) Lifor, both with active oxygenation (95%O2/5%CO2), as well as (3) Lifor passively oxygenated with ambient air (21%O2/0.04%CO2), all at ambient temperatures (20±2°C). At hypothermic temperatures (5±1°C) and under passive oxygenation were (4) University of Wisconsin solution (UW) and (5) Vasosol. Negative and positive control groups comprised livers that had ischemia (WI) and livers that did not (Fresh) prior to cell isolation, respectively. ResultsFresh livers yielded 32±9million cells/g liver while an hour of ischemia reduced the cell yield to 1.6±0.6million cells/g liver. Oxygenated Williams Medium E and Lifor recovered yields of 39±11 and 31±2.3million cells/g liver, respectively. The passively oxygenated groups produced 15±7 (Lifor), 13±7 (Vasosol), and 10±6 (UW)million cells/g liver. Oxygenated Williams Medium E was most effective at sustaining pH values, avoiding the accumulation of lactate, minimizing edematous weight gain and producing bile during perfusion. ConclusionsMachine perfusion results in a dramatic increase in cell yields from livers that have had up to an hour of warm ischemia, but perfusate choice significantly impacts the extent of recovery. Oxygenated Williams Medium E at room temperature is superior to Lifor, UW and Vasosol, largely facilitated by its high oxygen content and low viscosity.

High Mobility Group Box 1 Release from Hepatocytes during Ischemia and Reperfusion Injury Is Mediated by Decreased Histone Deacetylase Activity

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.

Reduced Ischemia-Reoxygenation Injury in Rat Intestine After Luminal Preservation With a Tailored Solution

The intestine is extremely sensitive to ischemic preservation and reoxygenation injury. Current vascular perfusion and cold storage with University of Wisconsin (UW) solution neglect the intestinal lumen and the ongoing mucosal metabolism during hypothermia. This study was designed to test the effects of luminal preservation with an alternative preservation solution in addition to the common vascular flush with UW solution on graft viability after preservation and ex vivo reoxygenation. Rat intestine was preserved on ice for 6 hr in UW solution or Williams Medium E with additional buffering, impermeants, and a colloid (WMEplus) after being stapled or after flushing and filling the lumen with the respective preservation solution. Tissue slices were prepared from fresh and preserved intestines and were incubated with oxygen for 6 hr at 37°C to assess the viability after reoxygenation. Directly after preservation, histologic damage was mild and unaffected by preservation strategy. Contrary to luminal preservation, closed preservation resulted in significantly decreased ATP levels compared with control. Reoxygenation aggravated damage and revealed differences between the strategies. Luminal preservation better maintained the ATP levels and histologic integrity (vs. closed preservation) for both solutions. Histomorphologic integrity was superior after preservation with WMEplus (vs. UW solution). Expression of stress responsive genes was least up-regulated in the slices from tissue preserved luminally with WMEplus. In conclusion, preservation and reoxygenation injury can be attenuated by luminal preservation with WMEplus.

Signal Transducer and Activator of Transcription 3 (STAT3) Mediates Amino Acid Inhibition of Insulin Signaling through Serine 727 Phosphorylation

Nutrient overload is associated with the development of obesity, insulin resistance, and type II diabetes. High plasma concentrations of amino acids have been found to correlate with insulin resistance. At the cellular level, excess amino acids impair insulin signaling, the mechanisms of which are not fully understood. Here, we report that STAT3 plays a key role in amino acid dampening of insulin signaling in hepatic cells. Excess amino acids inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis in mouse primary hepatocytes as well as in human hepatocarcinoma HepG2 cells. STAT3 knockdown protected insulin sensitivity from inhibition by amino acids. Amino acids stimulated the phosphorylation of STAT3 at Ser(727), but not Tyr(705). Replacement of the endogenous STAT3 with wild-type, but not S727A, recombinant STAT3 restored the ability of amino acids to inhibit insulin signaling, suggesting that Ser(727) phosphorylation was critical for STAT3-mediated amino acid effect. Furthermore, overexpression of STAT3-S727D was sufficient to inhibit insulin signaling in the absence of excess amino acids. Our results also indicated that mammalian target of rapamycin was likely responsible for the phosphorylation of STAT3 at Ser(727) in response to excess amino acids. Finally, we found that STAT3 activity and the expression of its target gene socs3, known to be involved in insulin resistance, were both stimulated by excess amino acids and inhibited by rapamycin. In conclusion, our study reveals STAT3 as a novel mediator of nutrient signals and identifies a Ser(727) phosphorylation-dependent and Tyr(705) phosphorylation-independent STAT3 activation mechanism in the modulation of insulin signaling.

Dual Mechanisms for the Fibrate-mediated Repression of Proprotein Convertase Subtilisin/Kexin Type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.

Antihepatotoxic and Antioxidant Activities of the Rwandan Botanicals, Ocimum lamiifolium and Crassocephalum vitellinum. Composition of their Essential Oils

The treatment of liver diseases with Rwandan botanicals occupies an important place in Rwandese traditional medicine and several plants or combinations of plants, among which Crassocephalum vitellinum and Ocimum lamiifolium leaves, are used by the traditional healers. The present work investigates the claimed antihepatotoxic and antioxidant activities of leave polar extracts, comparing to N-acetylcysteine (reference antihepatotoxicant) and both quercetin and Trolox (reference antioxidants). In order to evaluate the direct and protective effects of C. vitellinum and O. lamiifolium methanolic extracts on liver, rats precision cut liver slices (PCLS) were prepared and incubated in a Williams medium E with 1, 2, 5, 10 mg/ml of the extract either alone or in the presence of acetaminophen (hepatotoxicant, 10 mM). The measurement of ATP level and CYP2E1 activity were used as endpoints to assess liver toxicity and metabolic activity. Chlorzoxazone was used as a probe to assess CYP2E1 enzymatic activity and the metabolite 6-OH-chlorzoxazone formation was quantified by HPLC [1]. In addition to a significant hepatoprotective activity (1 mg/ml), the methanolic extracts of C. vitellinum and Ocimum lamiifolium leaves strongly scavenged the stable radical 1, 1-diphenyl-2-picryhydrazyl (DPPH) and reduced the peroxidation of linoleic acid. The essential oil was isolated by hydrodistillation (0.046% w/w yield for C. vitellinum and 0.077% w/w yied for O. lamiifolium) and analyzed with GC and GC/MS [2,3,4,5]. Identified predominating constituents of C. vitellinum oil are respectively limonene (34.8%), (E)-β-ocimene (21.8%), β-pinene (8.5%), α-pinene (6.6%),myrcene (6.3%) and β-phellandrene (5.5%) and the identified predominating constituents of O. lamiifolium oil are represented by sabinène (12.24%) and alpha phellandrène (11.62%).

High Circulating Leptin Receptors with Normal Leptin Sensitivity in Liver-specific Insulin Receptor Knock-out (LIRKO) Mice

Liver-specific insulin receptor knock-out (LIRKO) mice display hyperinsulinemia, abnormal glucose metabolism, and progressive liver dysfunction. In addition, circulating leptin levels appear to be increased more than 10-fold. However, food intake, body weight, and adipose mass are not significantly altered in LIRKO mice compared with wild-type littermates. Using a ligand immunofunctional assay, we found that the apparent increase in circulating leptin in LIRKO mice is because of an 80-fold increased serum level of soluble leptin receptor. Gene expression analysis by microarray and real time PCR reveals the liver as the source of soluble leptin receptor in LIRKO mice, with an increase in expression of the short (Ob-Ra), long (Ob-Rb), and soluble (Ob-Re) forms of the leptin receptor. Direct control of leptin receptor expression by insulin could also be demonstrated in isolated hepatocytes from normal mice. Despite the markedly increased levels of leptin receptor in their circulation, LIRKO mice exhibit normal or even enhanced leptin sensitivity, as assessed by their physiological and molecular responses to exogenous leptin administration and their lower base-line hypothalamic levels of SOCS3 mRNA. Thus, insulin signaling in the liver plays an important role in control of leptin receptor expression and shedding. In the LIRKO mouse, this is lost, leading to markedly increased leptin receptors into the circulation. These high levels of circulating leptin receptor bind leptin and likely alter its clearance, but do not inhibit leptin action and may actually potentiate leptin action. In this manner, insulin signaling in liver plays an important role in leptin homeostasis and fine modulation of leptin action.

Free radicals scavenging and hepatoprotective activities of the rwandese medicinal herb, Crassocephalum vitellinum and composition of the essential oil

The treatment of liver diseases occupies an important place in the Rwandese traditional medicine and several plants or combinations of plants, among which are Crassocephalum vitellinum leaves, are used by the traditional healers. The present work investigates the claimed antihepatotoxic and antioxidant activities of leaves polar extracts, comparing to N-acetyl cysteine (reference antihepatotoxicant) and both quercetin and Trolox (reference antioxidants). In order to evaluate the direct and protective effects of C. vitellinum methanolic extract on liver, rats precision cut liver slices (PCLS) were prepared and incubated in a Williams medium E with 1, 2, 5, 10mg/ml of the extract either alone or in the presence of acetaminophen (hepatotoxicant, 10mM). The measurement of ATP level and CYP2E1 activity were used as endpoints to assess liver toxicity and metabolic activity. Chlorzoxazone was used as a probe to assess CYP2E1 enzymatic activity and the metabolite 6-OH-chlorzoxazone formation was quantified by HPLC [1]. In addition to a significant hepatoprotective activity (1mg/ml), the methanolic extract of C. vitellinum leaves strongly scavenged the stable radical 1, 1-diphenyl-2-picryhydrazyl (DPPH) and reduced the peroxidation of linoleic acid. The essential oil was isolated by hydrodistillation (0,046% v/w yield) and analyzed with GC and GC/MS [2]. Identified predominating constituents that represent 95.8% of the total oil profile are limonene (34.8%), (E)-β-ocimene (21.8%), β-pinene (8.5%), α-pinene (6.6%), myrcene (6.3%), β-phellandrene (5.5%), germacrene-D (4.04%), α-phellandrene (3.6%),terpinolene (1.8%), Sabinene (1.7%), and β-caryophyllene (1.14%).

Effect of TRB3 on Insulin and Nutrient-stimulated Hepatic p70 S6 Kinase Activity

Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway.

Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy

Scavenger receptor class B type I (SR-BI) plays a critical role in the delivery of HDL cholesterol and cholesteryl esters (CEs) to liver and steroidogenic tissues by a selective process that does not result in significant degradation of HDL protein. Recently, SR-BI-mediated endocytosis and recycling of HDL have been demonstrated. However, it remains unclear whether efficient SR-BI-mediated selective uptake occurs strictly at the plasma membrane or at additional sites along its endocytic itinerary. To examine the requirement for SR-BI endocytosis in HDL selective uptake, we determined the effects of energy depletion on the levels of cell-associated HDL protein and CE in primary mouse hepatocytes. Compared with CHO cells, we observed a much larger energy-dependent effect on CE uptake in primary mouse hepatocytes. Although varying the levels of caveolin-1 and carboxyl ester lipase altered the efficiency of selective uptake, neither was able to account for the energy-dependent component of HDL-CE uptake. Finally, we demonstrate that the hepatocyte-specific, energy-dependent effects on HDL-apolipoprotein A-I and -CE uptake are independent of SR-BI and are not required to achieve efficient SR-BI-mediated selective uptake of CE. Together, these data support the conclusion that neither the intracellular trafficking of HDL nor any energy-dependent cellular process affects the ability of the cell to maximally acquire CE through SR-BI-mediated selective uptake from HDL.

42 PRODUCTION OF CLONED PIGS FROM SOMATIC STEM CELLS DERIVED FROM SALIVARY GLAND

The aim of the present study was to investigate whether a somatic stem cell derived from the salivary gland can be an efficient donor cell for pig cloning. Somatic stem cells (salivary gland progenitor cells, SGP) were isolated from the salivary gland of a 4-month old male pig (Matsumoto et al. 2004). Briefly, tissue sections of salivary gland were gently digested by collagenase/hyaluronidase and dispersed into single cells. Isolated cells (5 × 104) were cultured on a type I collagen-coated dish in William's medium E; then colonies having epithelial-like morphology were picked to establish the primary culture of SGP cells (CD49, intracellular laminin-positive) which have the potential to differentiate in vitro into hepatic and pancreatic endocrine cells after spherical body formation in vitro. SGP cells to be used as nuclear donors were cultured for 2 days under serum starvation. Fetal fibroblast (FF) cells were used as control nuclear donors. IVM oocytes were obtained from abattoir ovaries and matured in NCSU23. Donor cells were fused with the enucleated recipient oocytes by a single DC pulse of 190 V/mm for 10 μs in 0.28 M mannitol + 0.15 mM MgSO4. Reconstructed embryos were electrically activated by DC pulse of 150 V/mm for 100 μs in 0.28 M mannitol + 0.05 mM CaCl2 + 0.1 mM MgSO4 at 1–1.5 h after the NT, followed by cytochalasin B treatment for 3 h. Development of the NT embryos was assessed in vitro by fixing and staining at either 2 h post NT or after culture for 7 days in NCSU23, or in vivo by transfer to the oviducts of estrus-synchronized recipient gilts. Incidence of premature chromosome condensation was similar regardless of donor cell type. Development of the NT embryos reconstructed with SGP to the blastocyst stage was significantly higher compared to that of the FF group (38/137, 27.7% vs. 19/168, 11.3%, respectively; P &lt; 0.05). Transfer of 278 cloned embryos reconstructed with SGP to two recipients resulted in the production of three live piglets. Production efficiency of piglets from the cloned embryos reconstructed with FF was 2/263. Based on the in vitro development of the reconstructed embryos, SGP is a promising nuclear donor cell for pig cloning; further transfer experiments are to be carried out. This study was supported by PROBRAIN.

Initial Blood Washout During Organ Procurement Determines Liver Injury and Function After Preservation and Reperfusion

Organ procurement is the first step toward effective liver preservation and comprises a thorough washout of blood components from the microvasculature. To study the efficacy of optimal blood washout of the liver, three groups were compared including low-pressure perfusion with UW-CSS (12 mmHg, group A), which is the routine method in clinical practice, high-pressure perfusion with UW-CSS (100 mmHg, group B) and low-pressure perfusion with modified UW solution (12 mmHg, group C). After procurement all livers were preserved in original UW-CSS for 0, 24 or 48 h, followed by reperfusion in oxygenated Williams Medium E for 24 h at 37 degrees C. Histology results of livers procured in group A, showed good hepatocyte viability but also remaining erythrocytes. However, injury parameters were high and ATP concentrations were low. No functional differences were found. Group B, high pressure, and group C, modified UW-CSS, both showed better results. High-pressure washout is preferable since the warm ischemia time during procurement is short. We propose to use high-pressure UW-CSS perfusion for the initial blood washout of the donor liver instead of the usually used low-pressure washout.

A modified medium that significantly improves the growth of human normal ovarian surface epithelial (OSE) cells in vitro

Approximately 90% of malignant ovarian tumours are epithelial and thought to arise from a single cell layer, the ovarian surface epithelium. In culture, human normal ovarian surface epithelial (OSE) cells have a very limited lifespan before they senesce, rarely progressing beyond 10 population doublings. This has restricted the use of normal OSE cells for studying the biology of ovarian surface epithelium and identifying molecular events that contribute to malignant transformation. We have investigated the conditions for culturing human, normal OSE cells in vitro using modified media. Culturing normal OSE cells in a modified medium (NOSE-CM) supplemented with epidermal growth factor, hydrocortisone, insulin and bovine pituitary extract led to significant improvements in the seeding and cloning efficiencies, overall cell growth and lifespan compared to culturing in a basic, nonsupplemented medium (BM) and previously used media (F-12 K medium and William's medium E). Cells cultured in NOSE-CM underwent, on an average, 19.0 population doublings (95% CI 16.3–21.7); cells cultured in BM underwent 0.43–3.52 population doublings over a similar time period. Growth curves established for different lines indicated that OSE cells continued to grow beyond passage 11 and up to passage 18 in NOSE-CM, but never beyond passage 7 when cultured in BM. It is likely that establishing optimal conditions for the growth of OSE cells in vitro will enable studies of the biological and genetic mechanisms of transformation in epithelial ovarian cancers.

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 μM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 μM pregnenolone 16α-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.

Ex vivo stimulation of renal transport of the cytostatic drugs methotrexate, cisplatin, topotecan (Hycamtin) and raltitrexed (Tomudex) by dexamethasone, T3 and EGF in intact human and rat kidney tissue and in human renal cell carcinoma.

Previous experiments have shown that both in vivo and in vitro pre-treatment with various hormones increases the renal transport capacity for weak organic acids, such as PAH, in rats. The aim of the present study was to test whether or not accumulation of the anticancer drugs methotrexate (MTX), cisplatin (CP), raltitrexed (Tomudex) and topotecan (Hycamtin) can be increased in intact, healthy rat and human renal cortical slices and in human renal cell carcinoma (RCC). Intact, healthy human tissue was obtained from tumour bearing kidneys of patients suffering from RCC. Experiments were intended as a new approach to overcome so-called multidrug resistance. Kidney tissue slices were incubated for 24 h in William's medium E containing various concentrations of dexamethasone, T(3), or EGF. Thereafter slices were placed in anticancer drug containing Cross-Taggart medium and the drug uptake into kidney tissue was measured for 2 h. In intact rat and human renal tissue slices, the uptake of p-aminohippurate (PAH = reference substance) increased significantly after incubation in dexamethasone containing medium (134% and 156%, respectively). There were no stimulating effects of either T(3) or EGF on PAH accumulation. On the other hand, only the accumulation of MTX, but not of CP, raltitrexed or topotecan, was significantly enhanced after hormone pre-treatment both in intact renal tissue and in RCC. A stimulation of renal PAH accumulation can be performed ex vivo, as reported previously, both in intact rat and human renal cortical slices and in RCC. Discrepancies between the effects of dexamethasone and T(3) or EGF indicate different modes of action of these substances at the cellular level. Unfortunately, with the exception of MTX, the uptake of anticancer drugs can not be stimulated effectively ex vivo in human RCC tissue by the substances used. Evidently the transport of these anticancer drugs out of the kidney cells is more effective than their uptake.