Comparative Evaluation of Volume and Homogeneity of Obturation with Four Different Obturation Systems Using Micro-Computed Tomography: An In vitro Study.

Aim: To evaluate and compare volume and homogeneity of the three different root canal obturation systems. Materials and Methods: Single-rooted premolar (n = 24) teeth samples were selected, and crowns were removed for standardization. Four groups are divided randomly as (n = 6), namely: For group I (single-cone gutta-percha obturation), group II (Beefill 2 in 1 obturation), group III (GuttaCore obturation), group IV (GuttaFlow bioseal obturation) and the root canal were subjected to prepare till X3 (protaper next) and subjected to micro-CT imaging. After completion of obturation, the image was taken by using micro-CT imaging. This is to evaluate the volume of filled obturation material in the canal space and the voided area sections, viz. the apical, middle, coronal, and third sections. Results: Group III (GuttaCore obturation) showed the least significant mean of the difference in relation to the volume of the canal obturation (81.148). The least mean significant difference in area of voids in the canal region for apical (0.00133), middle (0.00233), and coronal thirds (0.00533). The most statistically significant difference is in the apical and middle thirds root canal space. Conclusion: All the experimental groups showed significant differences in volume and voids in the obturation at three different levels, and the GuttaCore obturation systems occupied more of the volume with less voids in the prepared root canal space.

Comparative Evaluation of Antibacterial Efficacy, Molecular Docking of Ethanolic Extract of Blackseed, Seaweed and Calcium Hydroxide Intracanal Medicament with Enterococcus Faecalis Antigens.

Aim: To evaluate the inhibitory effect of ethanolic extract blackseed, seaweed, and calcium hydroxide intracanal medicament with Enterococcus faecalis biofilm. To study the binding interaction between the active components of blackseed and seaweed against the enterococcal surface protein of (E. faecalis) by molecular docking. Materials and Methods: The ethanolic extracts of blackseed and seaweed were prepared using the Soxhlet apparatus. They were divided into three groups, namely, |Group I: Calcium hydroxide, Group II: Blackseed, and Group III: Seaweed. The antibacterial activity of the three groups was detected employing various concentrations ranging from 250, 125, and 62.5 µg/ml and based on the zone of inhibition. The inhibitory potential of medicaments to inhibit E. faecalis growth at various stages and kinetics plate were assessed following biofilm architecture evaluation by crystal violet biofilm assay. With the Swissdock suite, the molecular docking procedure was carried out. PyMOL version 4.1.5 was the program used for visualization. Since enterococcal surface protein (Esp) is primarily involved in the formation of biofilms, it was chosen as the target protein of E. faecalis. Based on their chromatographic investigations, Group II Thymoquinone (TQ) and Group III Ledenoxide were chosen as ligands. Results: The percentage of inhibition of E. faecalis biofilm was analyzed as statistically significant observed within groups. On post-hoc analysis, significant differences were present between the groups (P < 0.05). Molecular docking reveals binding energies of thymoquinone (Group II) and ledenoxide (Group III) against the enterococcal surface protein of E. faecalis were -6.90 Kcal/mol and -6.44 Kcal/mol, respectively. Conclusion: Compared to seaweed, black seed extract exhibited higher antibacterial activity against the E. faecalis biofilm in microbial inhibition and molecular interaction.

Comparative Evaluation of Fracture Resistance of Fiber-Reinforced Composite and Alkasite Restoration in Class I Cavity.

Background and Aim: Tooth-colored restorative materials for the restoration of decayed posterior teeth continue to gain popularity both among dental practitioners and patients. These materials have undergone a number of improvements in recent years to enhance their physical properties and diversify their use as a restorative material relevant to clinical practice. The aim of this study was to evaluate and compare the fracture resistance of two such advanced restorative materials, namely EverX Posterior, a fiber-reinforced composite and Cention N, an alkasite material in a Class I Cavity. Materials and Methods: Forty intact, caries-free human maxillary premolar teeth extracted for orthodontic purposes were divided randomly into four groups of 10 teeth each. Group I were unprepared teeth (intact teeth); Group II were unrestored teeth with class I cavity; Group III were teeth restored with fiber-reinforced composite (EverX Posterior); and Group IV were teeth restored using alkasite material (Cention N). Fracture resistance was recorded for all samples using a universal testing machine. Results: Higher fracture resistance was recorded in intact teeth group followed by EverX Posterior, Cention N and unrestored teeth, respectively. The teeth restored with EverX Posterior showed higher mean fracture resistance to fractures than those restored with Cention N. Teeth restored with EverX Posterior showed no significant difference in mean fracture resistance from Intact teeth while restored teeth with Cention N and unrestored teeth did. Conclusion: Fracture resistance of EverX Posterior was comparable to that of the natural tooth and was higher as compared to Cention N.

Microwave assisted synthesis of heterometallic 3d-4f M4Ln complexes.

In this paper we describe the synthesis and magnetic properties of a series of 3d-4f complexes of general formula [M4Ln(OH)2(chp)4(SALOH)5(H2O)(MeCN)(Solv)] (Solv = MeOH, MeCN, H2O; chp stands for deprotonated 6-chloro-2-hydroxypyridine (C5H3ClNO), SALOH stands for monodeprotonated 3,5-ditert-butylsalicylic acid (C15H21O3)) obtained by a solvent-free microwave assisted synthesis method. The Ni(ii) complexes (Ni4Gd, Solv = MeOH; Ni4Dy, Solv = MeCN) are not SMMs in the absence of an applied dc field. The replacement of Ni(ii) by Co(ii) (Co4La, Solv = MeOH; Co4Gd, Solv = H2O; Co4Gd-MeCN, Solv = MeCN; Co4Tb, Solv = MeOH; Co4Dy, Solv = H2O) results in improved SMM properties.

Antimicrobial efficacy of liquorice against Enterococcus faecalis biofilms in various concentrations at time-dependent variables: An in vitro study.

INTRODUCTION: The purpose this study was to evaluate the inhibitory efficacy of liquorice at various concentrations against Enterococcus faecalis and their biofilms at time-dependent variables in 24 h, 48 h, 72 h, 120 h, and 168 h. MATERIALS AND METHODS: The antienterococcal activity of liquorice and calcium hydroxide was detected employing concentration ranging from 1-4 g and interpreted based on the zone of inhibition. The ability of liquorice to inhibit E. faecalis biofilms during the stages of growth kinetics on microtiter plate was assessed, and the biofilm architecture was evaluated by scanning electron microscope (SEM). RESULTS: Statistically significant antienterococcal was observed at 3 and 4 g of liquorice against 24 and 48 h on microtiter plates. This observation was also complimented by SEM studies of biofilm architecture cultivated in root canals. CONCLUSIONS: E. faecalis biofilms at 24 h and 48 h were highly susceptible to liquorice at concentration of 3 and 4 g.

Evaluation of salivary nitric oxide level in children with early childhood caries.

BACKGROUND: Nitric oxide (NO), a highly reactive radical, participates in the nonspecific natural defense mechanism of the oral cavity. The present study was attempted to evaluate the salivary NO levels in 4-5 year-old children with early childhood caries (ECC). The objective of the present study was to assess the salivary NO concentration in children with different caries activity. MATERIALS AND METHODS: The study included 120 healthy 4.5 year-old children and they were equally divided into three groups based on decayed, missing, filled surfaces (dmfs) score; forty caries-free children (control group), forty children with dmfs 1.5 (ECC group), and forty with dmfs ⩾6 (severe ECC group). Saliva collected was measured for NO concentration by Griess reaction method. The obtained data were analyzed by ANOVA and Pearson's correlation coefficient. RESULTS: The mean level of NO in the saliva of the control group was 51.2 ± 8.3457 and that of ECC and severe ECC were 47.1 ± 5.2614 and 33.625 ± 4.6942, respectively. The mean salivary NO concentration was significantly higher in healthy controls when compared to children with ECC and severe ECC. Moreover, a negative correlation (r = -0.6658) was observed between the salivary NO level and the mean dmfs, suggesting that as the salivary NO level decreases, the caries incidence increases. CONCLUSION: The obtained results support the antimicrobial activity of salivary NO and also suggest that an increase in NO production might contribute to lower the caries occurrence in children.

A truncated [Mn(III)12] tetrahedron from oxime-based [Mn(III)3O] building blocks.

The use of the novel pro-ligand H4L combining the complimentary phenolic oxime and diethanolamine moieties in one organic framework, results in the formation of the first example of a [Mn(III)12] truncated tetrahedron and an extremely rare example of a Mn cage conforming to an Archimedean solid.

Homeobox genes and down-stream transcription factor PPARγ in normal and pathological human placental development.

The placenta provides critical transport functions between the maternal and fetal circulations during intrauterine development. Formation of this interface is controlled by nuclear transcription factors including homeobox genes. Here we summarize current knowledge regarding the expression and function of homeobox genes in the placenta. We also describe the identification of target transcription factors including PPARγ, biological pathways regulated by homeobox genes and their role in placental development. The role of the nuclear receptor PPARγ, ligands and target genes in human placental development is also discussed. A better understanding of these pathways will improve our knowledge of placental cell biology and has the potential to reveal new molecular targets for the early detection and diagnosis of pregnancy complications including human fetal growth restriction.

Analysis of in vitro chemoprevention of genotoxic damage by phytochemicals, as single agents or as combinations.

Cancer chemoprevention with low-dose combinations of bioactive phytochemicals instead of single agents has been suggested to induce less toxicity and improve efficacy. In this study, we selected four plant food-based phytochemicals, viz. chlorogenic acid (CLA), pelargonidin (PEL), resveratrol (RES) and epigallocatechin gallate (EGCG) to evaluate the in vitro chemoprevention of genotoxic damage in HL-60 cells. These agents were tested either individually or as a combination at two concentrations (with a 10-fold difference) against the genotoxins mitomycin C (MMC), diepoxybutane (DEB) and patulin (PAT). Our preliminary ferric reducing antioxidant power (FRAP) assay demonstrated additive effects when PEL, CLA, RES and EGCG were combined. Results of the cytokinesis-block micronucleus test showed significant protection against genotoxic damage induced by PAT, DEB and MMC when CLA, PEL, RES and EGCG were tested individually. This protective effect of the phytochemicals was not concentration-related. Both low- and high-concentration combinations of CLA, PEL, RES and EGCG showed significant reducing effects on the frequencies of micronuclei induced by PAT, DEB and MMC. However, the micronucleus test did not provide indications of additive or synergistic effects with this combination of phytochemicals. In conclusion, the chemo-preventive effects of PEL, CLA, RES and EGCG against genotoxic damage induced by MMC, DEB and PAT are indicative of a 'saturation effect' when higher concentrations and combinations of these phytochemicals are used.

Aldosterone induces oxidative stress, oxidative DNA damage and NF-κB-activation in kidney tubule cells.

An increase of the mineralocorticoid aldosterone is induced by a stimulated renin-angiotensin system in a subgroup of hypertensive patients. Epidemiological studies find higher cancer mortality in hypertensive patients and an increased risk to develop kidney cancer. This work investigated the involvement of oxidants in the genotoxicity of aldosterone and on a potential activation of transcription factor nuclear factor-κB (NF-κB) in kidney tubule cells. Aldosterone, at concentrations as low as 1 nM caused a significant increase of DNA damage, as assessed by comet assay and micronucleus frequency test. Aldosterone also led to a dose-dependent activation of NF-κB. Time courses of DNA damage and NF-κB-activation showed that these effects already occurred after 5 and 3 min of aldosterone exposure, respectively, suggesting non-genomic events of the hormone. Antioxidants prevented aldosterone-induced DNA damage and NF-κB-activation, indicating the involvement of oxidants. In fact, aldosterone caused an increase in intracellular oxidant levels, and in particular of superoxide anions. As a consequence, increased levels of the oxidized DNA modification 7,8-dihydro-8-oxo-guanine were observed in aldosterone-treated kidney cells. Aldosterone-induced DNA damage and NF-κB-activation was dependent on the involvement of the mineralocorticoid receptor. The induction of oxidant-mediated genotoxic effects, and of a long-term activation of the potentially oncogenic cell signal NF-κB by aldosterone could contribute to the increased kidney cancer incidence in hypertensive patients.

Mineralocorticoid receptor-mediated DNA damage in kidneys of DOCA-salt hypertensive rats.

Epidemiological studies exploring the connection between hypertension and cancer demonstrate a higher cancer incidence, especially of kidney cancer, and a higher cancer mortality in hypertensive patients. Hormones elevated in hypertension, i.e., aldosterone and angiotensin II, which exert genotoxic effects in vitro, could contribute to carcinogenesis in hypertension. The present study was conducted to investigate the possible DNA-damaging effect of aldosterone receptor activation in vivo. Crl:CD (Sprague-Dawley) rats were treated for 6 wk with desoxycorticosterone acetate (DOCA) and salt to induce a mineralocorticoid-dependent hypertension. DOCA-salt treatment caused increased blood pressure (+26 mmHg) compared to untreated rats, elevated markers of kidney failure (up to 62-fold for Kim-1), and the induction of several proinflammatory genes and proteins (up to 2.6-fold for tissue MCP-1). The mineralocorticoid receptor (MR) antagonist spironolactone (MR IC(50) 24 nM) and the novel nonsteroidal antagonist BR-4628 (MR IC(50) 28 nM) decreased these damage markers. DOCA-salt treatment also caused 8.8-fold increased structural DNA damage, determined with the comet assay, double-strand breaks (3.5-fold), detected immunohistochemically, and oxidative stress. Furthermore, the oxidatively modified mutagenic DNA base 7,8-dihydro-8-oxo-guanine (8-oxodG), quantified by LC-MS/MS, was almost 2-fold higher in DOCA-salt-treated kidneys. Our results suggest a mutagenic potential of high mineralocorticoid levels, frequent in hypertensive individuals.

Decorin expression is decreased in human idiopathic fetal growth restriction.

Fetal growth restriction (FGR) is a clinically significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. Most cases of FGR are idiopathic and are associated with placental thrombosis. Previous studies suggest that proteoglycans, such as decorin, that contain the glycosaminoglycan dermatan sulfate are the principal anticoagulants in the normal placenta. The present study investigated decorin expression in placentas from pregnancies complicated by idiopathic FGR (n = 26) and gestation-matched controls (n = 27). Real-time polymerase chain reaction demonstrated significantly reduced decorin mRNA expression in FGR compared with control (1.52 +/- 0.14 v. 2.21 +/- 0.22, respectively; P < 0.01). Immunoblotting revealed decreased decorin protein (40 kDa) expression in FGR compared with controls (420.8 +/- 39.0 v. 690.1 +/- 42.2, respectively; n = 12 in each group; P = 0.0007). Immunohistochemistry demonstrated the presence of immunoreactive decorin protein in the placental villous stroma surrounding the fetal capillaries and a significant decrease in decorin protein presence in FGR compared with control (1.75 +/- 0.66 v. 2.98 +/- 1.12, respectively; n = 6 in each group; P < 0.01, t-test). This is the first study to demonstrate reduced decorin in idiopathic FGR, indicating a potentially significant role for decorin in the aetiology of placental thrombosis in idiopathic FGR.

Placental biglycan expression is decreased in human idiopathic fetal growth restriction.

Fetal growth restriction (FGR) is a leading cause of perinatal morbidity and mortality. The majority of FGR cases are idiopathic and are associated with placental insufficiency, which can result from placental thrombosis. Evidence suggests that Dermatan Sulfate (DS) is an important anti-coagulant in placentae of uncomplicated pregnancies. This study hypothesised that the expression of biglycan proteoglycan, a source of DS, is decreased in idiopathic FGR placentae compared with placentae from uncomplicated pregnancies. This study aimed to determine biglycan mRNA, protein expression and spatial distribution in idiopathic FGR placentae compared with the placentae from gestation-matched controls. Biglycan mRNA expression, protein expression and spatial distribution was determined in 26 idiopathic FGR-affected placentae and 27 placentae from gestation-matched controls (27-40 weeks gestation) using real-time PCR, immunoblotting and immunohistochemistry, respectively. Mean biglycan mRNA expression was significantly decreased in FGR placentae compared with control placentae (2.87 +/- 0.55, (n = 26) vs. 4.48 +/- 0.85, (n = 27); t-test p = 0.01). FGR placentae demonstrated a trend towards decrease in mean biglycan protein expression compared with control placentae (0.86 +/- 0.22 (n = 9, FGR) vs, 1.9 +/- 0.56 (n = 7, control) p = 0.07). Biglycan immunoreactivity was detected in endothelial cells and sub-endothelial cells of the perivascular region of fetal capillaries. Semi-quantitative analyses demonstrated a significant decrease in immunoreactive biglycan in FGR placentae compared with control placentae (51.1 +/- 19.3 vs, 500.7 +/- 223, n = 6, p < 0.001). This is the first study to demonstrate decreased biglycan expression in idiopathic FGR placentae compared to gestation-matched controls. Reduced biglycan expression may contribute to placental thrombosis within the fetal vasculature, and may contribute to the pathogenesis of idiopathic FGR.

Novel homeobox genes are differentially expressed in placental microvascular endothelial cells compared with macrovascular cells.

Angiogenesis is fundamental to normal placental development and aberrant angiogenesis contributes substantially to placental pathologies. The complex process of angiogenesis is regulated by transcription factors leading to the formation of endothelial cells that line the microvasculature. Homeobox genes are important transcription factors that regulate vascular development in embryonic and adult tissues. We have recently shown that placental homeobox genes HLX, DLX3, DLX4, MSX2 and GAX are expressed in placental endothelial cells. Hence, the novel homeobox genes TLX1, TLX2, TGIF, HEX, PHOX1, MEIS2, HOXB7, and LIM6 were detected that have not been reported in endothelial cells previously. Importantly, these homeobox genes have not been previously reported in placental endothelial cells and, with the exception of HEX, PHOX1 and HOXB7, have not been described in any other endothelial cell type. Reverse transcriptase PCR was performed on cDNA from freshly isolated placental microvascular endothelial cells (PLEC), and the human placental microvascular endothelial cell line HPEC. cDNAs prepared from control term placentae, human microvascular endothelial cells (HMVEC) and human umbilical vein macrovascular endothelial cells (HUVEC) were used as controls. PCR analyses showed that all novel homeobox genes tested were expressed by all endothelial cells types. Furthermore, real-time PCR analyses revealed that homeobox genes TLX1, TLX2 and PHOX1 relative mRNA expression levels were significantly decreased in HUVEC compared with microvascular endothelial cells, while the relative mRNA expression levels of MEIS2 and TGIF were significantly increased in macrovascular cells compared with microvascular endothelial cells. Thus we have identified novel homeobox genes in microvascular endothelial cells and have shown that homeobox genes are differentially expressed between micro- and macrovascular endothelial cells.

Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent trophoblast cell proliferation.

The cytokine colony stimulating factor-1 (CSF-1) is a key regulator of the proliferation, differentiation and activation of mononuclear phagocytes. CSF-1 also plays an important role in reproduction. CSF-1 is produced in the placenta and activates signal transduction pathways that significantly increase the proliferation of placental trophoblast cells in culture. The target genes activated by CSF-1 mediated signal transduction in the nucleus are not well understood. Here, we use placental trophoblast cells to investigate potential downstream effector genes of CSF-1. HLX1 is a homeobox gene that controls proliferation in embryonic cell types and haematopoietic cell lineages. We have shown HLX1 is expressed in placental trophoblast cells but its functional role in the placenta is unknown. Following CSF-1 stimulation, HLX1 mRNA expression was significantly increased in SGHPL-4 and HTR-8/SVNeo cultured trophoblast cells (p<0.001, n=3). siRNA-mediated reduction of HLX1 mRNA levels with four independent oligonucleotides (siRNAs) resulted in significantly decreased cell proliferation in both cell lines (p<0.001, n=4). When HLX1 mRNA levels were reduced in the presence of CSF-1 stimulation, proliferation remained significantly decreased (p<0.001, n=4) in both the cell lines. We have shown for the first time that a homeobox gene, HLX1, is a downstream effector gene of CSF-1, that HLX1 regulates placental cell proliferation and that CSF-1 acts, at least in part, through HLX1 to control cell proliferation.

Role of cytosolic liver fatty acid binding protein in hepatocellular oxidative stress: effect of dexamethasone and clofibrate treatment.

The presence of cysteine and methionine groups together with an ability to bind long-chain fatty acid (LCFA) oxidation products makes liver fatty acid binding protein (L-FABP) an attractive candidate against hepatocellular oxidative stress. In this report, we show that pharmacological treatment directed at modulating L-FABP level affected hepatocellular oxidant status. L-FABP expressing 1548-hepatoma cells, treated with dexamethasone or clofibrate, decreased and increased intracellular L-FABP levels, respectively. Oxidative stress was induced by H2O2 incubation or hypoxia-reoxygenation. The fluorescent marker, dichlorofluorescein (DCF), was employed to measure intracellular reactive oxygen species (ROS). Hepatocellular damage was assessed by lactate dehydrogenase (LDH) level. Dexamethasone treatment resulted in a significant increase in DCF fluorescence with higher LDH release compared to control cells. Clofibrate treatment, however, resulted in a significant decrease in both parameters (p<0.05). Drug treatments did not affect cytosolic activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), or catalase suggesting that the differences between treated and control cells may likely be associated with varying L-FABP levels. We conclude that L-FABP may act as an effective endogenous cytoprotectant against hepatocellular oxidative stress.

Benign fibrous histiocytoma of the sacrum--diagnostic difficulties facing this rare bone tumor.

Benign fibrous histiocytoma of bone is rare, and this is the first report of the tumor arising from the sacrum. The descriptive histopathological picture makes the diagnosis more difficult as it resembles many benign and malignant myxoid tumors. In this case report, the diagnostic difficulties involved and a review of the literature in order to describe the optimal management of this condition, have been presented. The clinical presentation of this 18-year-old girl was that of low back ache, radiating along the right posterior leg, of 1 month's duration. On the straight leg raising test, right-sided sciatic nerve compression was detected. Magnetic resonance imaging revealed a large tumor involving and destroying sacral segments S3-S4, homogeneous in intensity and extending into the vertebral canal and the presacral space. There was no enhancement with gadolinium contrast. Core needle biopsy revealed fibrous histiocytoma. Excision through a posterior midline incision was performed taking care to preserve the S1-S2 sacral segments and also the sacral nerves. Additional cauterization with phenol was performed. Postoperatively, the patient had significant improvement in pain with no major residual neurological deficit.

Membrane binding proteins are the major determinants for the hepatocellular transmembrane flux of long-chain fatty acids bound to albumin.

PURPOSE: The hepatic transmembrane flux of long-chain fatty acids (LCFA) occurs through passive and fatty acid transport protein facilitated processes from blood. The extent that these transport processes can be related to the unbound and protein-bound fractions of LCFA in blood is not clear. METHODS: We used hepatocyte suspensions, hepatoma monolayers, and perfused rat livers to quantitate the transport of purified [(3)H]palmitate ([(3)H]PA) and 12-(N-methyl)-N-[(7-nitrobenz-2oxa-1,3-diazol-4yl-)amino]octadecanoicacid (12-NBDS) from solutions with a constant unbound LCFA concentration with varying bovine serum albumin (BSA) concentrations and in the presence and absence of antisera raised against cytosolic liver fatty acid binding protein (L-FABP). RESULTS: In the absence of L-FABP antisera, using an unbound ligand concentration that was adjusted to remain constant at each BSA concentration, hepatocyte [(3)H]PA and 12-NBDS uptake rates increased linearly with an increase in BSA concentration (p < 0.0001). In the presence of L-FABP antisera, [(3)H]PA uptake showed a greater reduction in the presence of 100 muM BSA than 5 muM BSA. The calculated permeability surface area product (PS) confirmed that both unbound and bound fractions of LCFA contributed to the overall flux, but only the PS for the protein-bound fraction was reduced in the presence of L-FABP antisera. In situ rat liver perfusion studies showed that the only rate process for the disposition of [(3)H]PA in the liver inhibited by L-FABP antisera was that for influx, as defined by PS, and that it reduced PS in the perfused liver by 42%. CONCLUSION: These results suggest that, at physiological albumin concentrations, most of the LCFA uptake is mediated from that bound to albumin by a hepatocyte basolateral membrane transport protein, and uptake of unbound LCFA occurring by passive diffusion contributes a minor component.

Assessing the cellular transmembrane electrical potential difference on the hepatic uptake of palmitate.

Understanding the driving forces for the hepatic uptake of endogenous and exogenous substrates in isolated cells and organs is fundamental to describing the underlying hepatic physiology/pharmacology. In this study we investigated whether uptake of plasma protein-bound [3H]-palmitate across the hepatocyte wall is governed by the transmembrane electrical potential difference (PD). Uptake was studied in isolated hepatocytes and isolated perfused rat livers (IPL). Protein-binding and vasoactive properties of the different perfusates were determined using in vitro heptane/buffer partitioning studies and the multiple indicator dilution (MID) technique in the IPL, respectively. Altering hepatocyte PD by perfusate ion substitution resulted in either a substantial depolarization (-14 +/- 1 mV, n = 12, mean +/- S.E., substituting choline for Na+) or hyperpolarization (-46 +/- 3 mV, n = 12, mean +/- S.E., substituting nitrate for Cl-). Perfusate ion substitution also affected the equilibrium binding constant for the palmitate-albumin complex. IPL studies suggested that, other than with gluconate buffer, hepatic [3H]-palmitate extraction was not affected by the buffer used, implying PD was not a determinant of extraction. [3H]-Palmitate extraction was much lower (p < 0.05) when gluconate was substituted for Cl- ion. This work contrasts with that for the extraction of [3H]-alanine where hepatic extraction fraction was significantly reduced during depolarization. Changing the albumin concentration did not affect hepatocyte PD, and [3H]-palmitate clearance into isolated hepatocytes was not affected by the buffers used. MID studies with vascular and extravascular references revealed that, with the gluconate substituted buffer, the extravascular volume possibly increased the diffusional path length thus explaining reduced [3H]-palmitate extraction fraction in the IPL.

Effect of dexamethasone, 2-bromopalmitate and clofibrate on L-FABP mediated hepatoma proliferation.

Cytosolic liver fatty acid binding protein (L-FABP) is involved in many intracellular functions including cellular mitogenesis. We investigated the role of L-FABP and the plasma membrane liver fatty acid binding proteins (L-FABP(pm)) in the modulation of hepatoma growth and proliferation, hypothesizing that agents that affect either the content of, or ligand binding to, L-FABP would affect hepatocellular mitogenesis. L-FABP expressing 1548-rat hepatoma cells were treated with 0.5 microM dexamethasone or 500 microM clofibrate for 4 days to downregulate and upregulate L-FABP expression, respectively. The competitive inhibitor 2-bromopalmitate (BrPA, 600 microM) was used to inhibit ligand binding to L-FABP. The peripherally present plasma membrane fatty acid transporter was inactivated by treating cells with 1:50 rabbit antisera (FABP-Ab) raised against L-FABP. Western blot analysis was used to monitor L-FABP levels while [(3)H]-thymidine incorporation and growth curves were used to monitor hepatocellular proliferation. [(3)H]-Palmitate clearance studies were performed using monolayer cultures. Palmitate clearance in dexamethasone-, BrPA- and FABP-Ab-treated cells was significantly reduced when compared with control (P < 0.05), while clofibrate treatment moderately increased the rate. [(3)H]-Thymidine incorporation by dexamethasone- and BrPA-treated cells was significantly lower than control (P < 0.05), suggesting that hepatocellular proliferation was inhibited. Clofibrate treatment did not statistically affect growth rate. Lowering L-FABP using dexamethasone or interfering with its activity using BrPA significantly affected hepatocellular proliferation. This may be due to the non-availability of long-chain fatty acids or other intracellular mediators that are transported by L-FABP to the nucleus.