The circadian clock gene Bmal1 facilitates cisplatin-induced renal injury and hepatization.

Cisplatin is one of the most potent chemotherapy drugs to treat cancers, but its clinical application remains limited due to severe nephrotoxicity. Several approaches have been developed to minimize such side effects, notably including chronotherapy, a well-known strategy based on the circadian clock. However, the component of the circadian clock machinery that particularly responses to the cisplatin stimulation remains unknown, including its functions in cisplatin-induced renal injury. In our present study, we demonstrated that Bmal1, as a key clock gene, was induced by the cisplatin stimulation in the mouse kidney and cultured human HK-2 renal cells. Gain- and loss-of-function studies indicated that Bmal1 facilitated cisplatin-induced renal injury both in vivo and in vitro, by aggravating the cell apoptotic process. More importantly, RNA-seq analysis revealed that Bmal1 triggered the expression of hallmark genes involved in renal hepatization, a critical event accompanied by the injury. At the molecular level, Bmal1 activated the transcription of hepatization-associated genes through direct recruitment to the E-box motifs of their promoters. Our findings suggest that Bmal1, a pivotal mediator induced renal injury in response to cisplatin treatment, and the therapeutic intervention targeting Bmal1 in the kidney may be a promising strategy to minimize the toxic side-effects of cisplatin in its clinical applications.

Grape seed proanthocyanidins protect against streptozotocin­induced diabetic nephropathy by attenuating endoplasmic reticulum stress­induced apoptosis.

Diabetic nephropathy (DN) is by far the most common cause of end­stage renal disease (ESRD) in industrial countries, accounting for ~45% of all new ESRD cases in the United States. Grape seed proanthocyanidin extracts (GSPE) are powerful antioxidants, with an antioxidant ability 50­fold greater than that of vitamin E and 20­fold greater than that of vitamin C. The present study investigated whether GSPE can protect against streptozotocin (STZ)­induced DN and aimed to elucidate a possible mechanism. Male Sprague Dawley rats were randomly divided into three groups: Control group (N), diabetes mellitus group (DM) injected with 40 mg/kg STZ, and the GSPE treatment group (intragastric administration of 250 mg/kg/day GSPE for 16 weeks after diabetes was induced in the rats). Blood and kidney samples were collected after treatment. The renal pathological changes were determined with periodic acid­Schiff (PAS) staining, while the protein expression levels of glucose­regulated protein 78 (GRP78), phosphorylated­extracellular signal­regulated kinase (p­ERK) and Caspase­12 were determined by western blotting and immunohistochemical staining. Apoptosis was determined with a terminal deoxynucleotidyl transferase dUTP nick­end labeling (TUNEL) assay. Compared with the DM group, the GSPE group had no significant changes in the blood urea nitrogen (BUN) level and serum creatinine (Scr) level, but showed a significant decline in the renal index (RI) level and 24­h urinary albumin level (P<0.05). The histopathology results indicated very little pathological damage in the GSPE group. Compared with the DM group, the GSPE group had a significantly reduced number of TUNEL­positive cells (P<0.05), and the GSPE group had an obvious reduction in the protein expression of GRP78, p­ERK, and Caspase­12 (P<0.05). In this study, the results indicated that GSPE can protect renal function and attenuate endoplasmic reticulum stress­induced apoptosis via the Caspase­12 pathway in STZ­induced DN.

Yi Guan Jian decoction may enhance hepatic differentiation of bone marrow­derived mesenchymal stem cells via SDF­1 in vitro.

A previous study reported that Yi Guan Jian (YGJ) may increase the proliferation and differentiation of hepatic oval cells in a rat liver cirrhosis model. The aim of the present study was to investigate the effect and mechanism of action of YGJ on inducing hepatic differentiation in bone marrow­derived mesenchymal stem cells (BM­MSCs) via stromal­cell derived factor­1 (SDF­1). Murine BM­MSCs were isolated with whole bone marrow adherence, then identified by immunocytochemical staining and flow cytometry. Passage 2 cells were divided into 8 groups and their differentiation was induced by cell factors added to the medium, including hepatocyte growth factor (HGF), SDF­1 and YGJ. Each of the cell factors was used alone and any two or three of them were combined to establish different cell microenvironments in the different treatment groups. Albumin (ALB) was selected as a hepatocellular marker and cytokeratin­18 (CK­18) as a cholangiocellular marker. The protein and mRNA expression levels of ALB and CK­18 were used to determine the differentiation of BM­MSCs using immunocytochemical staining, western blotting and reverse transcription­quantitative polymerase chain reaction on days 7, 14, 21 and 28 during induction. The relative expression levels of ALB and CK­18 resulted in time­dependent increases in the groups supplemented only with HGF, SDF­1 or YGJ. Combination treatment of any two HGF, SDF­1 and YGJ led to a higher expression of ALB and CK­18 compared with only one cell factor treatment. Additionally, when all three were used in a combined treatment the expression levels of ALB and CK­18 occurred at an earlier time and was higher overall. Therefore, the present study suggested that YGJ had an effect on inducing hepatic differentiation in BM­MSCs via SDF­1 and may act in a synergistic manner with HGF and SDF­1.

Oncostatin M in William's E medium is suitable for initiation of hepatocyte differentiation in human induced pluripotent stem cells.

William's E (WE) is a suitable medium for the differentiation of human induced pluripotent stem (iPS) cells to the hepatocyte lineage. The aim of the present study was to investigate various growth factors in their ability to promote hepatocyte differentiation of iPS cells in WE medium. Human iPS 201B7 cells were cultured in WE medium supplemented with growth factors, and mRNA expression levels and promoter activities of α­fetoprotein (AFP) and albumin were examined by reverse transcription­quantitative polymerase chain reaction and luciferase assay, respectively. In addition, time course analysis of AFP mRNA expression was performed in 201B7 cells cultured in WE medium supplemented with oncostatin M. The results demonstrated that mRNA expression levels of AFP were significantly elevated by most growth factors tested as supplements in WE medium, except all­trans retinoic acid, compared with cells cultured in ReproFF (a medium that maintains pluripotency). The highest increase in AFP mRNA expression levels was observed by oncostatin M stimulation. Albumin mRNA expression levels were increased by all­trans retinoic acid and insulin­transferrin­selenium supplementation in WE medium compared with cells cultured in ReproFF. Oncostatin M supplementation significantly stimulated the promoter activity of the AFP gene, but no growth factor tested significantly stimulated the promoter activity of the albumin gene. By time course analysis, significant increase of AFP mRNA expression was observed on the sixth day post­stimulation, compared with cells cultured in WE medium alone. In conclusion, the present study demonstrated that oncostatin M supplementation in WE medium was sufficient to initiate hepatocyte differentiation in iPS cells.

Ectopic expression of amaranth seed storage albumin modulates photoassimilate transport and nutrient acquisition in sweetpotato.

Storage proteins in plants, because of high nutrient value, have been a subject of intensive investigation. These proteins are synthesized de novo in the cytoplasm and transported to the storage organelles where they serve as reservoir of energy and supplement of nitrogen during rapid growth and development. Sweetpotato is the seventh most important food crop worldwide, and has a significant contribution to the source of nutrition, albeit with low protein content. To determine the behaviour of seed storage proteins in non-native system, a seed albumin, AmA1, was overexpressed in sweetpotato with an additional aim of improving nutritional quality of tuber proteins. Introduction of AmA1 imparted an increase in protein and amino acid contents as well as the phytophenols. The proteometabolomics analysis revealed a rebalancing of the proteome, with no significant effects on the global metabolome profile of the transgenic tubers. Additionally, the slower degradation of starch and cellulose in transgenic tubers, led to increased post-harvest durability. Present study provides a new insight into the role of a seed storage protein in the modulation of photoassimilate movement and nutrient acquisition.

Effect of allogeneic umbilical cord mesenchymal stem cell transplantation in a rat model of hepatic cirrhosis.

OBJECTIVE: To determine the effects of human umbilical cord mesenchymal stem cell (UCMSC) transplantation, alone or in combination with tanshinone IIA (Tan IIA) on hepatic cirrhosis in rats. METHODS: A rat model of cirrhosis was established. Rats were divided into control, UCMSC, and UCSMC plus Tan IIA groups. Rats in the UCMSC group were injected via the tail vein with 0.2 mL Dil-labeled UCMSC suspension. Intraperitoneal Tan IIA injections (20 mg/kg) were started on the day of UCMSC transplantation in the UCMSC plus Tan IIA group, and continued for 7 consecutive days thereafter. Rats were sacrificed 1 day, 3 days, 1 month, and 3 months after transplantation and the numbers of Dil-labeled UCMSCs colonizing the liver were determined. Albumin (ALB) and alanine aminotransferase (ALT) levels were measured in venous blood, and mRNA and protein expression levels of human ALB and cytokeratin (CK)-18 in liver tissues were determined by reverse transcription-polymerase chain reaction and western blotting, respectively. RESULTS: Serum ALT levels were significantly lower and serum ALB levels significantly higher in rats in the UCMSC group compared with the control group (P < 0.05). Hepatic CK-18 and ALB mRNA and protein expression levels increased after transplantation, and were significantly higher in the UCMSC plus Tan IIA group compared with the UCMSC group (P < 0.05). CONCLUSION: Human UCMSCs transplanted into rats with liver cirrhosis can grow and differentiate into hepatocyte-like cells resulting in improved liver function in vivo. Tan IIA further influenced transplantation outcomes.

Leucine improves protein nutritional status and regulates hepatic lipid metabolism in calorie-restricted rats.

Several studies have highlighted the potential of leucine supplementation for the treatment of metabolic diseases including type 2 diabetes and obesity. Caloric restriction is a common approach to improve the health in diabetic and obese subjects. However, very few studies assessed the effects of leucine supplementation in calorie-restricted animals. Rats were subjected to a 30% calorie-restricted diet for 6 weeks to study the effects of leucine supplementation on protein status markers and lipid metabolism. Caloric restriction reduced the body weight. However, increased leucine intake preserved body lean mass and protein mass and improved protein anabolism as indicated by the increased circulating levels of albumin and insulin-like growth factor-1 (IGF-1), and the liver expression of albumin and IGF-1 messenger RNA. Leucine supplementation also increased the circulating levels of interleukin-6 and leptin but did not affect the tumour necrosis factor-α and monocyte chemotactic protein-1 concentrations. Ketone bodies were increased in rats consuming a leucine-rich diet, but we observed no changes in cholesterol or triglycerides concentrations. Caloric restriction reduced the liver expression of peroxisome proliferator activated receptor-α and glucose-6-phosphatase, whereas leucine supplementation increased the liver expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA) reductase and sterol regulatory element-binding transcription factor 1. A leucine-rich diet during caloric restriction preserved whole body protein mass and improved markers of protein anabolism. In addition, leucine modulated the hepatic lipid metabolism. These results indicate that increased leucine intake may be useful in preventing excessive protein waste in conditions of large weight loss.

Cold storage of rat hepatocyte spheroids.

Cell-based therapies for liver disease rely on a high-quality supply of hepatocytes and a means for storage during transportation from site of isolation to site of usage. Unfortunately, frozen cryopreservation is associated with unacceptable loss of hepatocyte viability after thawing. The purpose of this study was to optimize conditions for cold storage of rat hepatocyte spheroids without freezing. Rat hepatocytes were isolated by a two-step perfusion method; hepatocyte spheroids were formed during 48 h of rocked culture in serum-free medium (SFM). Spheroids were then maintained in rocked culture at 37 °C (control condition) or cold stored at 4 °C for 24 or 48 h in six different cold storage solutions: SFM alone; SFM + 1 mM deferoxamine (Def); SFM + 1 µM cyclosporin A (CsA); SFM + 1 mM Def + 1 µM CsA, University of Wisconsin (UW) solution alone, UW + 1 mM Def. Performance metrics after cold storage included viability, gene expression, albumin production, and functional activity of cytochrome P450 enzymes and urea cycle proteins. We observed that cold-induced injury was reduced significantly by the addition of the iron chelator (Def) to both SFM and UW solution. Performance metrics (ammonia detoxification, albumin production) of rat hepatocyte spheroids stored in SFM + Def for 24 h were significantly increased from SFM alone and approached those in control conditions, while performance metrics after cold storage in SFM alone or cold storage for 48 h were both significantly reduced. A serum-free medium supplemented with Def allowed hepatocyte spheroids to tolerate 24 h of cold storage with less than 10% loss in viability and functionality. Further research is warranted to optimize a solution for extended cold storage of hepatocyte spheroids.

Genetic diversity of storage proteins in cultivated buckwheat.

Prolamin and albumn variations of the storage proteins in 76 cultivated buckwheat accessions (55 accessions of Fagopyrum tataricum, 21 accessions of F. esculentum) from 7 countries were characterized by A-PAGE and SDS-PAGE, respectively, for the purpose of evaluating the genetic diversity of cultivated buckwheat at the level of proteins. A total of 18 prolamin bands were detected, among which 88.89 % bands were polymorphic. The number of albumn bands based on SDS-PAGE observed in accessions ranged from 4 to 10. Most intense bands were in the range of molecular weights from 29 to 97.2 kDa. The average of genetic similarity coefficient based on prolamin bands was 0.784 (in F. tataricum and F. esculentum were 0.892 and 0.681, respectively), while on prolamin and albumn bands was 0.742 (in F. tataricum and F. esculentum were 0.864 and 0.633, respectively). Accessions of F. tataricum and F. esculentum showed significant interspecific variation in the A-PAGE and SDS-PAGE profile of the storage proteins. The cluster analysis indicated that all the accessions could be divided into 3 groups and 3 subgroups. The genetic variations among cultivated buckwheat accessions were associated with their geographic origins in some degree.

Radioiodine therapy of hepatoma using targeted transfer of the human sodium/iodide symporter gene.

UNLABELLED: We investigated the feasibility of radioiodine therapy targeting hepatoma cells (MH3924A) by tissue-specific expression of the human sodium/iodide symporter (hNIS) gene directed by the murine albumin enhancer and promoter (mAlb). METHODS: The cell-specific transcriptional activity of mAlb was examined by a luciferase assay in several transiently transfected cell lines. MH3924A cells were stably transfected with the recombinant retroviral vector, in which hNIS complementary DNA expression was driven by mAlb and coupled to hygromycin resistance gene using an internal ribosomal entry site (IRES). Functional hNIS expression in hepatoma cells was confirmed by an iodide uptake assay. In imaging studies, the tumor-bearing ACI rats were intravenously injected with (131)I and imaged with a gamma-camera. Biodistribution was studied at 30 min and at 1, 3, 6, and 25 h after injection of (131)I. Toxic effects of (131)I on hepatoma cells were studied in vitro and in vivo. RESULTS: Stably transfected MH3924A cells concentrated (125)I up to 240-fold higher than the wild-type cells. The iodide uptake in stably transfected cells was inhibited by ouabain and sodium perchlorate but increased by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. An in vitro clonogenic assay revealed an 86% decrease in colony number in stably transfected cells after exposure to 3.7 MBq/mL of (131)I and only about 8% in hNIS-negative control cells. Furthermore, the in vivo study showed intense tracer accumulation in hNIS-expressing tumors after administration of (131)I. At 3 h after intraperitoneal injection, the transfected tumors accumulated (131)I 19.2-fold higher than the parental tumors in a biodistribution study. Moreover, administration of a therapeutic dose of (131)I resulted in an inhibition of hNIS-expressing tumor growth, whereas control tumors continued to increase in size. CONCLUSION: A therapeutic effect of (131)I on hepatoma cells in vitro and in vivo has been demonstrated after tumor-specific iodide uptake induced by mAlb-directed hNIS gene expression. Because a stable transformed cell line has been used in these experiments, the clinical potential of this strategy must be evaluated after in vivo transfection of hepatoma cells.

Soybean oil in total parenteral nutrition maintains albumin and antioxidant enzyme mRNA levels.

Changes in albumin and antioxidant enzyme mRNA expression in infant rat liver following administration of total parenteral nutrition (TPN) with/without soybean oil emulsion were studied. Infant rats were divided into three groups: group 1=oral diet, group 2=TPN without fat, and group 3=TPN with 20% of calories from soybean oil emulsion. The period of TPN administration was 4 d. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in group 2 than in the other groups, with similar levels seen in the other groups. Albumin, Cu, Zn-superoxide dismutase, and glutaredoxin 1 mRNA expression levels were lower in group 2 than in the other groups, with similar levels seen in the other groups. Catalase mRNA expression was higher in group 1 than in the other groups, with the lowest level seen in group 2. Soybean oil emulsion should be included in TPN regimens to prevent down-regulation of albumin and antioxidant enzyme mRNA expression.

Pretreatment of starved rats with ornithine alpha-ketoglutarate: effects on hepatic mRNA levels and plasma concentrations of three liver-secreted proteins.

OBJECTIVE: Ornithine alpha-ketoglutarate (OKG) displays anabolic properties at the hepatic level, but the mechanisms involved remain unclear. This study investigated in vivo the ability of OKG to modulate hepatic gene expression of three liver-secreted proteins: albumin, transthyretin, and retinol binding protein. METHODS: One hundred eighty rats were fed for 5 d with a balanced regimen enriched with OKG (5 g.kg(-1).d(-1)) or an isonitrogenous mixture (alanine, glycine, and serine). Hepatic mRNA levels and plasma concentrations of the three proteins studied were determined at the end of the nutrition period and after 1, 2, and 3 d of food deprivation. Results were compared by analysis of variance and Bonferroni-Dunn tests. RESULTS: At the end of the nutrition period, hepatic mRNA levels and plasma concentrations of the three proteins were not modified by OKG supplementation. However, OKG largely increased mRNA levels of albumin, transthyretin, and retinol binding protein on the first day of starvation compared with control animals (+68%, +64% and +51%, respectively; P < 0.01 versus control). OKG precociously increased albuminemia (on day 2) but had no effect on plasma concentrations of transthyretin and retinol binding protein. Neither regulation of polyamine hepatic concentration nor alteration in hepatic amino acid content seemed to be implicated in these actions. CONCLUSION: This study is the first to demonstrate that OKG regulates in vivo liver gene expression during acute malnutrition by modulating hepatic mRNA levels.

Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.

OBJECTIVES: The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-L-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. METHODS: Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. RESULTS: Human pancreatic cell (HPC) cultures coexpressed alpha-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. CONCLUSION: These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.

Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function.

Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA([Ser]Sec) and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function.

Hypothalamic gene expression in reproductively photoresponsive and photorefractory Siberian hamsters.

An interval timing mechanism in the brain governs reproduction in seasonally breeding mammals by triggering refractoriness to inhibitory short photoperiods during midwinter. The neural mechanisms responsible for the timing and induction of photorefractoriness by this seasonal clock are unknown. Using cDNA microarrays and RT-PCR, we identified a class of genes encoding thyroxine (T4)-binding proteins (transthyretin, T4-binding globulin, albumin) whose expression is associated with reproductive refractoriness to short day lengths. Down-regulation of these genes was associated with reduced hypothalamic T4 uptake, which was reversed by long-day photoperiod treatments that restored responsiveness to short days. Circulating T4 concentrations did not vary with states of photoresponsiveness in euthyroid hamsters, but blockade of thyroid function accelerated the onset of photorefractoriness to short days. These data link changes in gene expression in the hypothalamus to the functional output of a seasonal clock. Reproductive inhibition in short days depends on T4 only late in the nonbreeding season. Down-regulation of genes encoding T4-binding proteins in the hypothalamus during this interval may restrict access of a static T4 signal to hypothalamic target tissues that regulate reproduction, thereby timing annual transitions in reproductive function. Hypothalamic autoregulation of T4 influx may constitute a critical cellular process involved in the generation and expression of seasonal reproductive rhythms and suggests a previously undescribed mechanism by which neural targets gain access to peripheral hormones.

Effect of the molar ratio of branched-chain to aromatic amino acids on growth and albumin mRNA expression of human liver cancer cell lines in a serum-free medium.

Supplementation of branched-chain amino acids (BCAAs) is often used for the treatment of hepatic encephalopathy and low albuminemia in Japan. In this scenario, although many cases are complicated with hepatocellular carcinoma in chronic viral infection, the effect of BCAA levels on hepatocellular carcinoma cells remains unclear. We investigated the effect of the molar ratios of BCAAs to aromatic amino acids (AAAs) on the growth and albumin mRNA expression of cultured human liver cancer cell lines, HCC-M, HCC-T, PLC/PRF/5, and Hep G2. To exclude the effect of fetal serum in culture media on modification of the growth and albumin transcription of cell lines, we used a synthetic serum-free medium. We found that an increase in the molar ratio of BCAAs to AAAs reduced the growth of Hep G2 cells, and it increased albumin mRNA expression in this cell line at a molar ratio of 0.1-10. These results suggest that the molar ratio of BCAAs to AAAs affect the growth and mRNA expression of some liver cancer cells, and supplementation of BCAAs may at least be beneficial to patients with cirrhosis, even complicated with liver cancer.

[Study on effect of nephropathy mixture on genetic expression of liver albumin in rats with adriamycin-induced nephrotic syndrome].

OBJECTIVE: To explore the effect of Nephropathy mixture on genetic expression of liver albunin in rats with adriamycin-induced nephrotic syndrome. METHODS: The rat model of adriamycin-induced nephrotic syndrome was established and the liver albumin mRNA expression level was observed with Northern hybridization and Dot-blot quantitative analysis. RESULTS: The liver albumin mRNA expression level in the Nephropathy Mixture treated group was significantly higher than that in the model group and the normal control group (P < 0.05). CONCLUSION: Nephropathy Mixture could up-regulate the liver albumin mRNA expression level and promote the synthesis of albumin in rats with adriamycin-induced nephrotic syndrome.

Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus.

Improvement of nutritive value of crop plants, in particular the amino acid composition, has been a major long-term goal of plant breeding programs. Toward this end, we reported earlier the cloning of the seed albumin gene AmA1 from Amaranthus hypochondriacus. The AmA1 protein is nonallergenic in nature and is rich in all essential amino acids, and the composition corresponds well with the World Health Organization standards for optimal human nutrition. In an attempt to improve the nutritional value of potato, the AmA1 coding sequence was successfully introduced and expressed in tuber-specific and constitutive manner. There was a striking increase in the growth and production of tubers in transgenic populations and also of the total protein content with an increase in most essential amino acids. The expressed protein was localized in the cytoplasm as well as in the vacuole of transgenic tubers. Thus we have been able to use a seed albumin gene with a well-balanced amino acid composition as a donor protein to develop a transgenic crop plant. The results document, in addition to successful nutritional improvement of potato tubers, the feasibility of genetically modifying other crop plants with novel seed protein composition.

Establishment and cellular characteristics of a hepatocyte cell line (OUMS-31) derived from an acatalasemic mouse.

Liver cell lines with very low catalase activity were established from an acatalasemic mouse. Hepatocytes isolated by a collagenase-liver-perfusion technique were cultured in Williams' E medium supplemented with 10% fetal bovine serum. The acatalasemic liver cell line showed approximately 20% of the catalase activity of a normal mouse liver cell line, whereas its glutathione peroxidase activity was approximately equal to that of the normal liver cell line. DNA sequence analysis of this cell line showed the same mutation in the catalase gene as is seen in the acatalasemic mouse. Our observation of intracellular content of hydrogen peroxide (H2O2) radical and increased susceptibility of the cells to H2O2 were compatible with the existence of low catalase activity in the acatalasemic mouse. This hepatocyte cell line should be useful for studying effects of oxidative radical stress at the cellular level.

Overexpression of C/EBPbeta represses human papillomavirus type 18 upstream regulatory region activity in HeLa cells by interfering with the binding of TATA-binding protein.

The human papillomavirus type 18 (HPV-18) upstream regulatory region (URR) controls cell type-specific expression of viral oncoproteins E6 and E7. The HPV-18 URR is highly active in HeLa cells, but its activity is virtually undetectable in HepG2 cells. Previous work has shown that YY1 plays an important role in activation of the HPV-18 URR in HeLa cells, and this activating activity is dependent on its physical interaction with C/EBPbeta, which binds to the switch region adjacent to the YY1 site in the URR. Overexpression of C/EBPbeta in HepG2 cells restores C/EBPbeta-YY1 interaction, resulting in strong activation of the HPV-18 URR activity. In this report, we show that, in contrast to the effect in HepG2 cells, overexpression of C/EBPbeta represses the HPV-18 URR in HeLa cells. This C/EBPbeta-induced repression of the HPV-18 URR in HeLa cells is binding site independent. It is also promoter specific, since it activates the albumin promoter under conditions in which it represses the URR in the same cells. Biochemical analysis shows that overexpression of C/EBPbeta in HeLa cells specifically interferes with binding of TATA-binding protein to the TATA box of the HPV-18 URR, but its overexpression in HepG2 cells leads to activation of the HPV-18 URR. These results suggest that a molecular mechanism underlies the ability of C/EBPbeta to regulate transcription in a cell type-specific manner and indicate the potential of using C/EBPbeta to manipulate the activity of the HPV-18 URR in cervical carcinoma cells.