High Responsiveness to Thyroid Hormone of Adult Rat Primary Hepatocytes Cultured on EHS-Gel

The induction of malic enzyme gene expression by triiodothyronine and insulin was severely blunted in rat monolayer hepatocytes cultured on type I collagen compared with that in spherical hepatocytes cultured on a reconstituted basement membrane gel (EHS-gel). Although the mRNA level of thyroid hormone receptor β (TRβ) gradually decreased in the monolayer hepatocytes during culture, the mRNA level in the hepatocytes on EHS-gel was maintained at around the in vivo level. Our results suggest that the maintenance of TRβ mRNA on EHS-gel is responsible for the high responsiveness to thyroid hormone in a hepatocyte culture.     

Modulation of cytochrome P450 gene expression in primary hepatocytes on various artificial extracellular matrices

We studied effect of artificial extracellular matrices (ECMs), such as collagen I, poly (N-p-vinylbenzyl-4-O-β-d-galactopyranosyl-d-gluconamide)(PVLA) and E-cadherin–IgG Fc (E-cad-Fc) on hepatic metabolism to identify the mechanism of in vivo hepatocellular functional and metabolic integrity. mRNA expression of liver function marker, cytochrome P450 (CYP) and transporter genes in hepatocytes were compared among used ECMs using real-time RT-PCR. mRNA expressions of Cyp2c29 and Cyp2d22 among CYP genes in hepatocytes on PVLA were recovered after 3 days due to enhanced liver-specific function by the spheroid formation of hepatocytes whereas mRNA expressions of CYP genes in hepatocytes on collagen and E-cad-Fc drastically decreased with time. mRNA expressions of the Cyp2c29 and Cyp2d22 in hepatocytes on PVLA were more recovered in the presence of epidermal growth factor (EGF) due to the more and bigger spheroid formation of hepatocytes. Multidrug resistance-associated protein 2 (Mrp2) protein was accumulated at intracellular lumen as similar to bile duct in hepatocyte spheroid formed on PVLA, indicating that spheroid formation of hepatocytes is very important for maintaining liver functions.     

Role of E-cadherin Molecules in Spheroid Formation of Hepatocytes Adhered on Galactose-Carrying Polymer as an Artificial Asialoglycoprotein Model

The role of E-cadherin in the spheroid formation of hepatocytes adhered on the poly(N-p-vinylbenzyl-D-lactonamide) (PVLA) as a model ligand for asialoglycoprotein receptors (ASGP-R) of hepatocytes was studied. Expression of E-cadherin was increased in round hepatocytes adhered on a high-coating density of PVLA (100 μg/ml), and also in flat ones adhered on a low-coating density of PVLA (1 μg/ml) in the presence of epidermal growth factor (EGF). Hepatocyte spheroids formed on the high-coating density of PVLA in the presence of EGF after 48 h were inhibited by an anti-E-cadherin monoclonal antibody (ECCD-1). From immunofluorescence and confocal laser microscopy, E-cadherin was localized in the intercellular boundaries and concentrated at the inside surface of aggregated cells. As a result, E-cadherin could play an important role in hepatocyte assembly.     

A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Low asialoglycoprotein receptor expression as markers for highly proliferative potential hepatocytes.

Development of a reliable method to isolate highly proliferative potential hepatocytes will provide insight into the molecular mechanisms of liver regeneration, as well as proving crucial for the development of a biohybrid artificial liver. The aim of this study is to isolate highly proliferative, e.g., progenitor-like, hepatocytes. To this end, we fractionated hepatocytes expressing low and high levels of the asialoglycoprotein receptor (ASGP-R) based on the difference in their adhesion to poly[N-p-vinylbenzyl-O-beta-d-galactopyranosyl-(1-->4)-d-gluconamide] (PVLA), and examined the proliferative activity and gene expression of these fractionated hepatocytes. The results showed that approximately 0.5 to 1% of the total number of hepatocytes, which showed low adhesion to PVLA, expressed low levels of the ASGP-R, while the rest of hepatocyte population with high adhesion to PVLA expressed high levels of the ASGP-R. Interestingly hepatocytes with low ASGP-R expression levels had much higher DNA synthesizing activity (i.e., are much more proliferative) than those with high ASGP-R expression levels. Moreover, hepatocytes with low ASGP-R expression levels expressed higher levels of epidermal growth factor receptor (EGF-R), CD29 (beta1 integrin) and CD49f (alpha6 integrin) and lower levels of glutamine synthetase than those with high ASGP-R expression. These findings suggested that hepatocytes with low adhesion to PVLA due to their low ASGP-R expression could be potential candidates for progenitor-like hepatocytes due to their high proliferative capacity; hence, the low expression of the ASGP-R could be a unique marker for progenitor hepatocytes. The isolation of hepatocytes with different functional phenotypes using PVLA may provide a new research tool for a better understanding of the biology of hepatocytes and the mechanisms regulating their proliferation and differentiation in health and disease.     

Terthieno[3,2‐b]Thiophene (6T) Based Low Bandgap Fused‐Ring Electron Acceptor for Highly Efficient Solar Cells with a High Short‐Circuit Current Density and Low Open‐Circuit Voltage Loss

A terthieno[3,2‐b]thiophene ( 6T ) based fused‐ring low bandgap electron acceptor, 6TIC , is designed and synthesized for highly efficient nonfullerene solar cells. The chemical, optical, and physical properties, device characteristics, and film morphology of 6TIC are intensively studied. 6TIC shows a narrow bandgap with band edge reaching 905 nm due to the electron‐rich π‐conjugated 6T core and reduced resonance stabilization energy. The rigid, π‐conjugated 6T also offers lower reorganization energy to facilitate very low V_OC loss in the 6TIC system. The analysis of film morphology shows that PTB7‐Th and 6TIC can form crystalline domains and a bicontinuous network. These domains are enlarged when thermal annealing is applied. Consequently, the device based on PTB7‐Th : 6TIC exhibits a high power conversion efficiency (PCE) of 11.07% with a high J_SC > 20 mA cm^?2 and a high V_OC of 0.83 V with a relatively low V_OC loss (≈0.55 V). Moreover, a semitransparent solar cell based on PTB7‐Th : 6TIC exhibits a relatively high PCE (7.62%). The device can have combined high PCE and high J_SC is quite rare for organic solar cells.     

TIME FOR COFFEE encodes a nuclear regulator in the Arabidopsis thaliana circadian clock

The plant circadian clock is required for daily anticipation of the diurnal environment. Mutation in Arabidopsis thaliana TIME FOR COFFEE (TIC) affects free-running circadian rhythms. To investigate how TIC functions within the circadian system, we introduced markers for the evening and morning phases of the clock into tic and measured evident rhythms. The phases of evening clock genes in tic were all advanced under light/dark cycles without major expression level defects. With regard to morning-acting genes, we unexpectedly found that TIC has a closer relationship with LATE ELONGATED HYPOCOTYL (LHY) than with CIRCADIAN CLOCK ASSOCIATED1, as tic has a specific LHY expression level defect. Epistasis analysis demonstrated that there were no clear rhythms in double mutants of tic and evening-acting clock genes, although double mutants of tic and morning-acting genes exhibited a similar free-running period as tic. We isolated TIC and found that its mRNA expression is continuously present over the diurnal cycle, and the encoded protein appears to be strictly localized to the nucleus. Neither its abundance nor its cellular distribution was found to be clock regulated. We suggest that TIC encodes a nucleus-acting clock regulator working close to the central oscillator.     

Regulation of Iron Homeostasis in Arabidopsis thaliana by the Clock Regulator Time for Coffee

In plants, iron homeostasis is tightly regulated to supply sufficient amounts of this metal for an optimal growth while preventing excess accumulation to avoid oxidative stress. To identify new regulators of iron homeostasis, a luciferase-based genetic screen using the Arabidopsis AtFer1 ferritin promoter as a target was developed. This screen identified TIME FOR COFFEE (TIC) as a regulator of AtFer1 gene expression. TIC was previously described as a nuclear regulator of the circadian clock. Mutants in the TIC gene exhibited a chlorotic phenotype rescued by exogenous iron addition and are hypersensitive to iron during the early stages of development. We showed that iron overload-responsive genes are regulated by TIC and by the central oscillator of the circadian clock. TIC represses their expression under low iron conditions, and its activity requires light and light/dark cycles. Regarding AtFer1, this repression is independent of the previously characterized cis-acting element iron-dependent regulatory sequence, known to be involved in AtFer1 repression. These results showed that the regulation of iron homeostasis in plants is a major output of the TIC- and central oscillator-dependent signaling pathways.     

The regulatory role of vernalization in the expression of low-temperature-induced genes in wheat and rye

Low temperature is one of the primary stresses limiting the growth and productivity of wheat (Triticum aestivum L.) and rye (Secale cereale L.). Winter cereals low-temperature-acclimate when exposed to temperatures colder than 10°C. However, they gradually lose their ability to tolerate below-freezing temperatures when they are maintained for long periods of time in the optimum range for low-temperature acclimation. The overwinter decline in low-temperature response has been attributed to an inability of cereals to maintain low-temperature-tolerance genes in an up-regulated state once vernalization saturation has been achieved. In the present study, the low-temperature-induced Wcs120 gene family was used to investigate the relationship between low-temperature gene expression and vernalization response at the molecular level in wheat and rye. The level and duration of gene expression determined the degree of low-temperature tolerance, and the vernalization genes were identified as the key factor responsible for the duration of expression of low-temperature-induced genes. Spring-habit cultivars that did not have a vernalization response were unable to maintain low-temperature-induced genes in an up-regulated condition when exposed to 4°C. Consequently, they were unable to achieve the same levels of low-temperature tolerance as winter-habit cultivars. A close association between the point of vernalization saturation and the start of a decline in the Wcs120 gene-family mRNA level and protein accumulation in plants maintained at 4°C indicated that vernalization genes have a regulatory influence over low-temperature gene expression in winter cereals.     

Comparative genomics of Lbx loci reveals conservation of identical Lbx ohnologs in bony vertebrates

Background  

Lbx/ladybird genes originated as part of the metazoan cluster of Nk homeobox genes. In all animals investigated so far, both the protostome genes and the vertebrate Lbx1 genes were found to play crucial roles in neural and muscle development. Recently however, additional Lbx genes with divergent expression patterns were discovered in amniotes. Early in the evolution of vertebrates, two rounds of whole genome duplication are thought to have occurred, during which 4 Lbx genes were generated. Which of these genes were maintained in extant vertebrates, and how these genes and their functions evolved, is not known.     

Long-distance signals positively regulate the expression of iron uptake genes in tobacco roots

Long-distance signals generated in shoots are thought to be associated with the regulation of iron uptake from roots; however, the signaling mechanism is still unknown. To elucidate whether the signal regulates iron uptake genes in roots positively or negatively, we analyzed the expressions of two representative iron uptake genes: NtIRT1 and NtFRO1 in tobacco (Nicotiana tabacum L.) roots, after shoots were manipulated in vitro. When iron-deficient leaves were treated with Fe(II)-EDTA, the expressions of both genes were significantly reduced; nevertheless iron concentration in the roots maintained a similar level to that in roots grown under iron-deficient conditions. Next, all leaves from tobacco plants grown under the iron-deficient condition were excised. The expression of two genes were quickly reduced below half within 2 h after the leaf excision and gradually disappeared by the end of a 24-h period. The NtIRT1 expression was compared among the plants whose leaves were cut off in various patterns. The expression increased in proportion to the dry weight of iron-deficient leaves, although no relation was observed between the gene expression and the position of excised leaves. Interestingly, the NtIRT1 expression in hairy roots increased under the iron-deficient condition, suggesting that roots also have the signaling mechanism of iron status as well as shoots. Taken together, these results indicate that the long-distance signal generated in iron-deficient tissues including roots is a major factor in positive regulation of the expression of NtIRT1 and NtFRO1 in roots, and that the strength of the signal depends on the size of plants.     

Translocons on the inner and outer envelopes of chloroplasts share similar evolutionary origin in Arabidopsis thaliana

The process of importing nuclear encoded proteins into chloroplasts is mediated by the T ranslocons on the O uter/I nner Envelope of C hloroplasts (TOC and TIC complex). The ancestor of the TOC complex was formed by pre‐existing proteins from the cyanobacterial ancestor; other proteins recruited from the host cell or cyanobacterial ancestor were subsequently integrated into the complex. However, little is known about the origin of the TIC complex. In this work, the origin of the TIC complex was investigated through one of its channel proteins, AtTic21. Phylogenetic analysis suggested that AtTic21 is conserved in photosynthetic organisms. AtTic21 showed 33% sequence identity to a Synechocystis protein SynTic21. The successful genetic complementation of an AtTic21 knockout mutant by SynTic21 plus the transit peptide coding sequence of AtTic21 suggested that SynTic21 is an ortholog of AtTic21. The sequence and functional conservation between SynTic21 and AtTic21 suggested that the TIC complex shares a similar evolutionary origin to the TOC complex.     

Mos1 transposition as a tool to engineer the Caenorhabditis elegans genome by homologous recombination

Gene knockouts and knock-ins have emerged as powerful tools to study gene function in model organisms. The construction of such engineered alleles requires that homologous recombination between a transgenic fragment carrying the modifications desired in the genome and the locus to engineer occurs at high frequencies. Homologous recombination frequency is significantly increased in the vicinity of a DNA double-strand break. Based on this observation, a new generation of transgene-instructed genome engineering protocols was developed. Here, we present MosTIC (for “Mos1 excision-induced transgene-instructed gene conversion”), a new technique that provides a means to engineer the Caenorhabditis elegans genome. MosTIC is initiated by the mobilization of Mos1, a Drosophila transposon experimentally introduced in C. elegans. During MosTIC, a Mos1 insertion localized in the genomic region to engineer is mobilized after germline expression of the Mos transposase. Mos1 excision generates a DNA double-strand break, which is repaired by homologous recombination using a transgenic repair template. This results in the transfer of information from the transgene into the genome. Depending on the method used to trigger Mos1 excision, two alternative MosTIC protocols are available, which are presented here in detail. This technique can be used for a wide range of applications, such as structure-function analysis, protein localization and purification, genetic screens or generation of single copy transgenes at a defined locus in the genome.     

Gene expression modulation by chalcopyrite and bornite in Acidithiobacillus ferrooxidans

Acidithiobacillus ferrooxidans is a mesophilic, acidophilic, chemolithoautotrophic bacterium that obtains energy from the oxidation of ferrous iron (Fe²⁺), elemental sulfur and reduced sulfur compounds. The industrial interest in A. ferrooxidans resides in its capacity to oxidize insoluble metal sulfides into soluble metal sulfates, thus allowing the recovery of the desired metals from low-grade sulfide ores. In the present work, RNA arbitrarily primed PCR (RAP-PCR) was performed to identify cDNAs differentially expressed in A. ferrooxidans cells grown in the presence of Fe²⁺ and cells maintained for 24 h in the presence of the copper sulfides bornite and chalcopyrite. Eighteen cDNAs corresponding to genes with known function were identified, and their relative expression was further characterized by real-time quantitative PCR. Bornite had a mild effect on the expression of the 18 genes analyzed. None of these genes was down-regulated and among the few genes up-regulated, it is worth mentioning lepA and def-2 that are involved in protein synthesis. Chalcopyrite presented the most significant changes. Five genes related to protein processing were down-regulated, and another 5 genes related to the transport system were up-regulated. The up- and down-regulation of these genes in the presence of bornite and chalcopyrite could be due to alterations in the ideal pH, presence of copper ions in solution and nutrient limitation. The results suggest that gene expression modulation might be important for the A. ferrooxidans early response to copper sulfides.     

Regulation of Harvest-induced Senescence in Broccoli (Brassica oleracea var. italica) by Cytokinin, Ethylene, and Sucrose

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene by accelerating the process, and cytokinin by delaying it. To determine the level at which these hormones influenced senescence, we isolated and monitored the expression of genes normally associated with senescence in broccoli florets treated with exogenous 6-benzyl aminopurine (6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC), a combination of 6-BAP and ACC, and sucrose, in the five days following harvest. Exogenous 6-BAP caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression. The expression of genes used as senescence markers, BoCP5 and BoMT1, was reduced, whereas BoCAB1 levels were maintained after harvest in response to exogenous 6-BAP. In addition, the expression of genes encoding sucrose transporters (BoSUC1 and BoSUC2) and carbohydrate metabolizing enzymes (BoINV1 and BoHK1) was also reduced upon 6-BAP feeding. Interestingly, the addition of ACC prevented the 6-BAP-induced increase in expression of BoACS, but 6-BAP negated the ACC-induced increase in expression of BoACO. The culmination of these results indicates a significant role for cytokinin in the delay of senescence. The implication that cytokinin regulates postharvest senescence in broccoli by inhibiting ethylene perception and/or biosynthesis, thus regulating carbohydrate transport and metabolism, as well as senescence-associated gene expression, is discussed and a model presented.     

The Mechanism of Hematopoietic Progenitor Cell immortalization by MLL-ENL

The t(11;19) translocation gives rise to the MLL-ENL fusion protein and is frequently found in infant myeloid and lymphoid leukemias. Immortalized myeloid cell lines can be generated by expression of MLL-ENL in murine hematopoietic progenitors. By establishing myeloid cell lines with conditional expression of MLL-ENL, we recently demonstrated that MLL-ENL is necessary to maintain immortalization and sustain the expression of a characteristic pattern of Hox genes. The cell lines can be induced to undergo terminal differentiation by inhibition of MLL-ENL expression or by treatment with G-CSF. Expression of Hoxa genes is reduced in cells differentiating as a result of MLL-ENL loss, but is maintained in G-CSF treated cells. Thus, although aberrant maintenance of Hoxa gene expression may play an important role in MLL-ENL induced leukemia, the contribution of this pathway to immortalization is critically dependent on the cytokine environment of the immortalized myeloid cells.     

Caffeine treatment of ovine cytoplasts regulates gene expression and foetal development of embryos produced by somatic cell nuclear transfer

Treatment of ovine oocytes during the latter stages of maturation in vitro with caffeine, a phosphodiesterase inhibitor, can increase the activities of maturation promoting factor and mitogen‐activated protein kinases at metaphase II. When used as cytoplast recipients for somatic cell nuclear transfer (NT), caffeine‐treated oocytes produced blastocysts with increased cell numbers. The objectives of these studies were to determine the effects of caffeine treatment on the expression profile of genes involved in early embryonic development and whether induction or maintenance of pregnancy was subsequently altered. No differences in overall expression patterns were observed between fertilised, caffeine‐treated fertilised and parthenogenetic embryos. In control NT embryos, altered levels of gene expression were found for OCT4, five genes regulated by OCT4 (H2AF.Z, NANOG, SOX2, FGF4 and INFT) and the heat‐shock response genes (HSP27 and HSP70.1). Levels of OCT4, H2AF.Z, NANOG, HSP 27 and FGF4 decreased, while those of INFT, HSP70.1 and SOX2 increased. In contrast, expression levels of these genes in caffeine‐treated NT embryos were similar to those in fertilised controls. Following transfer to surrogate recipients no differences were observed in the frequency of pregnancy; however, ewes receiving caffeine‐treated embryos maintained pregnancies for longer periods and delivered a live lamb. Taken together, these results suggest that treatment of ovine oocytes with caffeine can affect gene expression and improve developmental competence. Further studies on the mechanisms behind this alteration of gene expression are required and will aid in understanding the molecular mechanisms involved in nuclear reprogramming. Mol. Reprod. Dev. 77:876–887, 2010. © 2010 Wiley‐Liss, Inc.