Determination of potent antisense oligonucleotides in vitro by semiempirical rules

The selection of effective antisense target sites on a given mRNA molecule is a major problem in the detection of target mRNA in oligonucleotide arrays. In general, antisense oligodeoxynucleotides (asODNs) of about 10-20 nucleotides (nt) in length are used. However, the demand for predicting the sequence of potent asODNs much longer than those mentioned above has been increasing. Here, we prepared 40-nt asODNs directed against fluorescence-labeled green fluorescent protein (GFP) mRNA and quantified their hybridization efficiencies by fluorescence microscopy. We found that the hybridization efficiency depended on the TC content or the minimum free energy of the asODNs. On the basis of these findings, a semiempirical parameter called accessibility score was introduced to predict the potency of asODNs. The results of this study aided in the development of an effective two-step procedure for determining mRNA accessibility, namely, the computer-aided selection of asODN binding sites using an accessibility score followed by an experimental procedure for measuring the hybridization efficiencies between the selected asODNs and the target mRNA by fluorescence microscopy.     

Suppression of cell death in primary rat hepatocytes by alpha1-acid glycoprotein

In screening for effective additives for the long-term culture of hepatocytes, the hepatoprotective effect of alpha1-acid glycoprotein (AGP) was observed. AGP prevented primary hepatocytes from undergoing cell death induced by the chemical toxin, bromobenzene. Moreover, AGP added to medium was found to maintain the number of viable hepatocytes for as long as 6 d. The hepatoprotective effect of AGP was lost by removing sialic acid groups at the N-glycan chain terminal of AGP. It is shown that the complete form of N-glycan chain is needed for the hepatoprotectivity of AGP.     

Albumin production activity of primary rat hepatocytes is improved on type V collagen

Primary rat hepatocytes were cultured on type V collagen. Hepatocyte spreading on type V collagen was inferior compared with that on type I collagen. However, the albumin production rates of hepatocytes cultured on type V collagen were approximately twice as high as those of hepatocytes cultured on type I collagen.     

In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells

A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.     

Effects of the mycotoxin deoxynivalenol on human primary hepatocytes

Toxic effects of the mycotoxin deoxynivalenol (DON) observed in animals range from diarrhea, vomiting, gastro-intestinal inflammation to necrosis of several tissues. In the last years, DON has been tested in hepatocytes of several animal species for its cytotoxicity. However, these tests are limited to the use of animal cells. No studies using human hepatocytes are available. Further investigations with the human hepatocellular liver carcinoma cell line HepG2 might be limited due to the disadvantages of cell lines (e. g. immortalization, tumor derivation, longtime cultivation) and do not necessarily reflect the response of normal human cells. In order to overcome this problem and to be closer to the human situation, we studied the effect of DON in human primary hepatocytes and compared these data to the effects in the HepG2 cell line. Cell viability, apoptotic and necrotic cell death, albumin secretion and metabolic activity were determined. It could be demonstrated that DON has a distinct cytotoxic effect on human primary hepatocytes. Viability, protein content and albumin secretion were reduced in a dose-dependent manner. The apoptotic key enzyme caspase-3 was activated, while LDH release occurred only after long incubation time due to a secondary necrosis. Furthermore, we studied the metabolism of DON using LC-MS/MS. DON was neither metabolized by primary hepatocytes cells nor by the HepG2 cell line.     

Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH)

We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.     

Promotion of monolayer formation and high expression of ammonia metabolism of primary rat hepatocytes on arginine-glycine-aspartic acid-containing peptide-coated polystyrene dish

Cell adhesive peptide Arg-Gly-Asp (RGD) was immobilized using ProNectin F (PnF) on a nontreated polystyrene petri dish at a PnF density of 20 ng cm(-2), which is sufficient for primary rat hepatocyte immobilization. The density of PnF on the dish affects cell morphology and expression of the differentiated functions within the range of 2-2500 ng cm(-2). An optimal monolayer state with defined cell boundaries and hepatocyte nuclei was formed on a 2500 ng cm(-2) PnF-coated petri dish, and the ammonia metabolic function was expressed at as high a level as in the hepatocyte/spheroid. We conclude that 2500 ng cm(-2) PnF enhances the morphological stability and expression of liver-specific functions of the hepatocyte.     

Effect of sugar residues in a glycolipid coated onto a dish on ammonia consumption and gluconeogenesis activity of primary rat hepatocytes

Coating of 3,4,5-tris(dodecyloxy)benzyl-beta-D-galactopyranoside (TDOB-Gal) on a dish for suspension culture increased the specific ammonia consumption rate of primary rat hepatocytes to 2.4 times that in the case of rat hepatocytes cultured in a dish without coating while there was no increase in the specific urea production rate. TDOB-beta-D-glucopyranoside (TDOB-Glc), -alpha-D-mannnoside, -beta-D-mannoside, 2-acetamido-2-deoxy-beta-D-glucopyranoside, and 2-acetamido-2-deoxy-beta-D-galactopyranoside had almost no influence on the above-mentioned specific rates. In the ammonia loading assay, cells on the dish with TDOB-Gal and -Glc coatings produced glucose, suggesting gluconeogenesis.     

Micropatterned organoid culture of rat hepatocytes and HepG2 cells

The culture of liver cell organoids (multicellular aggregates) such as spheroids or cylindroids, which can strongly express liver functions, has been advocated as a useful technique that has advantages over monolayer culture. This paper describes a micropatterning technique for obtaining spheroids and cylindroids by using rat hepatocytes or HepG2 cells. We developed culture chips that comprised multiple, circular or rectangular microwells; the bottom surface of each microwell was modified with collagen to create a cell adhesion area, and the entire microwell, excluding the collagen-coated spots, was modified with polyethylene glycol (PEG) to create a nonadhesive area. Rat hepatocytes and HepG2 cells formed uniform spheroids and cylindroids on the circular and rectangular chips, respectively. Consequently, two-dimensional micropatterned chips containing homogeneous spheroids or cylindroids were generated. The expression of liver functions (protein secretion and ammonia removal) was greater in the spheroids and cylindroids than in the monolayer culture, and this expression was maintained for at least 2 weeks of culture. Thus, this chip technology has potential for use in various applications that involve organoid culture.     

The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells

Heterospheroids consisting of hepatocytes and bone marrow cells (BMCs) are formed by the mixed coculture of these cells and enhance the expression and maintenance of the liver-specific functions of hepatocytes. Not only the soluble factors derived from these cells, but also functional organoid (heterospheroid) formation, are considered to underlie this coculture effect. Therefore, in the present study, we aimed to clarify the mechanism of this co-culture effect. We performed hepatocyte monoculture with conditioned media prepared from hepatocyte cultures, BMC cultures and a coculture of hepatocytes and BMCs. When using any type of conditioned medium, no hepatocyte spheroids formed, and the hepatocytes formed a monolayer. In addition, an effect for these conditioned media was shown in terms of the albumin production and ammonia metabolism activities of the hepatocytes; conditioned medium from BMCs showed the strongest effect. The monocultured hepatocytes in the conditioned medium derived from BMCs showed equivalent albumin production and ammonia metabolism activities to the cocultured spheroids of hepatocytes and BMCs. Therefore, it was determined that the effect of the coculture of hepatocytes and BMCs was caused by soluble factors derived from BMCs.     

Novel function of rare catechin, epigallocatechin-3-(3''-O-methyl)gallate, against cold injury in primary rat hepatocytes

Epigallocatechin-3-(3'-O-methyl)gallate (EGCg-3'-OMe) is a rare component in green tea leaf and its bioactivity is hardly known. In this paper, we report that EGCg-3'-OMe has the function for cold preservation of primary rat hepatocytes. Confluent primary cultured hepatocytes were suspended in a storage solution, culture medium or cell banker (CB). EGCg-3'-OMe was tested as a supplement in the storage solution together with a general cryoprotectant, dimethylsulfoxide (DMSO). After 24 h cold preservation of cells at 4 degrees C followed by 1 h rewarming, cell viability and urea-synthesizing activity, one of the most important liver functions, were measured. EGCg-3'-OMe dose-dependently maintained cell viability and this effect was equal to that of a commercial CB at the highest concentration. Cell viability was also maintained after a further 24 h incubation at 37 degrees C of the cold-preserved hepatocytes. Conversely, urea-synthesizing activity was dose-dependently reduced by EGCg-3'-OMe. Cell protection by EGCg-3'-OMe due to the decrease in metabolic activity in cold-preserved cells was suggested. The decreased hepatic function of cells caused by EGCg-3'-OMe was rescued after a further 24 h incubation of cells at 37 degrees C.     

Cytoprotective effect of a bilberry extract against oxidative damage of rat hepatocytes

The effect of a bilberry extract (BE, 25% anthocyanins) against oxidative damage in primary cultures of rat hepatocytes, induced by tert-butyl hydroperoxide and allyl alcohol, was investigated. BE displayed cytoprotective effects at 100 and 500 μg/ml in the MTT viability test. It protected the cells against lactate dehydrogenase leakage and lipoperoxidation products formation. Maximum protection (58%) was noted using 500 μg/ml of BE and intoxication by allyl alcohol. The observed cytoprotective effect is probably due to the antioxidant properties of its constituents, mainly anthocyanins. BE scavenged DPPH (IC₅₀ 3.99 ± 0.14 μg/ml) and enzymatically generated superoxide radical with an activity equivalent to 108 ± 7.2 U of superoxide dismutase per mg of extract. Our results support the use of bilberry and bilberry extracts in functional foods and food supplements designed for the prevention of chronic diseases associated with oxidative stress.     

Cultivation of rat hepatocytes in a medium based on commercially available infusate solutions

The possibility of using commercially available infusate solutions as a culture medium for hepatocytes was investigated in primary monolayer cultures of rat hepatocytes. The addition of Ca2+ to the infusate medium was necessary for hepatocytes to express their albumin secreting ability. The infusate medium supplemented with hormones (10(-7) M insulin and 10(-7) M dexamethasone) and Ca2+ (72.5 mg/l) allowed hepatocytes to produce albumin of an amount comparable to that produced in Williams' E medium. The activity of released lactate dehydrogenase (LDH) was kept at a low level throughout the cultivation in the infusate medium.     

Administration of rosemary essential oil enhances resistance of rat hepatocytes against DNA-damaging oxidative agents

The aim of this paper was to evaluate the potential DNA-protective effects of rosemary essential oil-supplementation on rat hepatocytes damaged with three different genotoxins attacking DNA by oxidative stress. Hydrogen peroxide (H₂O₂) reacts by the generation of hydroxyl radicals, visible light-excited methylene blue forms oxidative DNA lesions via singlet oxygen and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) induces oxidative stress by participation in redox cycling. Hepatocytes were isolated from rats supplemented with rosemary oil (RO) for 14 days as well as from control rats. The potential protective effects of RO applied in drinking water of animals were tested on the level of DNA using the conventional and modified single cell gel electrophoresis (comet assay). We found out that administration to rats of rosemary oil, exhibiting free radical-scavenging activity measured by DPPH assay, efficiently and significantly decreased the level of DNA damage induced with H₂O₂, visible light-excited methylene blue and DMNQ.     

Growth factor/heparin-immobilized collagen gel system enhances viability of transplanted hepatocytes and induces angiogenesis

Hepatocyte transplantation is being explored as a treatment strategy for end-stage liver disease; however, the main limitation is the insufficient vascularization of transplanted hepatocytes. To overcome this problem, a suitable 3D microenvironment and the types of transplanted cells must be considered for hepatocyte transplantation. In this study, a growth factor (GF)/heparin-immobilized collagen gel-filled polyurethane foam (PUF) scaffold was developed for angiogenesis induction and hepatocyte transplantation. First, a vascular endothelial growth factor (VEGF)/heparin-immobilized, collagen-gel-filled PUF scaffold was developed to establish a prevascularized cavity in the subcutaneous space in rats. Second, accompanied by 70% partial hepatectomy (PH), hepatocytes were embedded inside heparin-immobilized, collagen-gel-filled PUF scaffolds, and were transplanted into the VEGF-induced prevascularized cavity. The benefits of using this system were confirmed by using three types of hepatocytes, namely single hepatocyte, hepatocyte spheroids, and fetal hepatocytes. The normalized hemoglobin content and live nucleus numbers were determined separately to evaluate the angiogenesis and viability of transplanted hepatocytes. In summary, after PH pretreatment, transplantation of fetal hepatocyte-embedded, heparin-immobilized, collagen-gel-filled PUF scaffold into a VEGF-induced prevascularized cavity appears to be a promising strategy for future liver tissue engineering.     

Bacteriophage resistance in Lactococcus lactis ssp. lactis using antisense ribonucleic acid.

Antisense RNA against a conserved bacteriophage gene when expressed in a Lactococcus lactis ssp. lactis strain renders it resistant to bacteriophage infection. Two open reading frames have been identified in a L. lactis ssp. lactis bacteriophage that are conserved in a majority of isolates. They code for an 18-kDa (designated GP18C) protein and a 24-kDa (GP24C) protein, respectively, which are arranged along with previously identified open reading frames in a tandem motif similar to other bacteriophages. The presence of gp18C and gp24C in a number of bacteriophage isolates was confirmed by polymerase chain reaction using primers specific for these regions. Plasmids bearing various fragments of gp18C, gp24C, or both were constructed such that the respective open reading frames were positioned in the antisense direction relative to the Lactococcus lactis ssp. cremoris Wg2 promoter, p59. These antisense RNA-producing vectors inhibited the efficiency of plaquing of L. lactis ssp. lactis bacteriophage phi 7-9 up to 50%; the resulting plaques were extremely small and irregular in shape. The replication of the bacteriophage was severely inhibited, and the total number decreased over the first 3 h during infection in strains expressing antisense RNA compared with the host strain alone, in which the bacteriophage number increased 10(4)-fold.