Mutation of the start codon to enhance Cripavirus internal ribosome entry site-mediated translation in a wheat germ extract.

Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5' terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3-4-fold higher than that of its wild type, and also 3-4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE.

In vitro selection of a 3' terminal short protector that stabilizes transcripts to improve the translation efficiency in a wheat germ extract.

Wheat cell-free expression systems based on wheat germ extract (WGE) enable us to briefly synthesize various types of proteins in vitro merely by exogenously adding their mRNA templates. Moreover, it is possible to produce larger amounts of protein by thoroughly removing the endosperm, which contains many translation inhibitors, including ribonucleases (RNases). However, because small amounts of RNases are also present even in an endosperm-free, high-quality WGE (hqWGE), the in-vitro transcribed mRNA is rapidly degraded. In particular, 3' exonucleases have been considered as the major RNases that degrade mRNA. We thus herein performed in vitro selection to find an effective, short 3' protector sequence from a random RNA pool. The selected sequences stabilized in vitro transcripts in the hqWGE more effectively than the previously reported, longer 3' protectors did. In addition, when one of these 3' protectors was minimized and then fused to mRNA, the translation efficiency increased 5-6-fold in the hqWGE, mainly due to the mRNA stabilization.

Biofunction-assisted DNA detection through RNase H-enhanced 3' processing of a premature tRNA probe in a wheat germ extract.

We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3' premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3' end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity.

Polymyositis associated with autoimmune hepatitis, primary biliary cirrhosis, and autoimmune thrombocytopenic purpura.

We describe a 40-year-old woman with polymyositis (PM) who developed autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), and autoimmune thrombocytopenic purpura (AITP) concurrently. About 4 years earlier, she suffered from muscle weakness probably due to PM. When she visited our hospital, she had polyarthritis, myalgia, symmetrical proximal limb-muscle weakness, elevated muscle enzymes, and myogenic abnormalities on electromyogram. Pathological findings obtained by muscle biopsy showed histological findings consistent with PM. Her serum liver enzymes were also elevated. The histology obtained by liver biopsy revealed the mixture findings of chronic active hepatitis and biliary cirrhosis. As antibodies to mitochondria M2 and liver/kidney microsome type 1 (LKM-1) were present, we concluded her liver disease was due to an overlap of AIH and PBC. Furthermore, purpura on the legs with thrombocytopenia appeared in parallel with liver dysfunction. She was diagnosed as having AITP by clinical and laboratory findings. Her serum showed a speckled pattern in immunofluorescence antinuclear antibody testing, but the antigen specificities were distinct from those of the known myositis-related autoantigens. This is a first case report of PM accompanied by AIH, PBC, and AITP. It was notable that there was an overlap of disease-associated immunological findings and immunogenetic backgrounds. This case provides a possible insight into the mechanisms and interplay of autoimmune diseases.

[The experiences of elderly patients' successful resumption of oral ingestion with home parenteral nutrition and multi-professional team approach].

There are many elderly patients with poor oral intake when they are hospitalized with pneumonia or urinary tract infection, and we often need to consider their proper feeding method, such as percutaneous endoscopic gastrostomy(PEG)or total parenteral nutrition(TPN). However, it is difficult to receive homecare services for patients who rely highly upon medical treatment. Meanwhile, a prolonged hospitalization is a serious social problem. Here, we report two cases of elderly patients who were able to eat because home parenteral nutrition care and seamless approach were provided by multi-professional team.

Generation of novel genetically modified rats to reveal the molecular mechanisms of vitamin D actions.

Recent studies have suggested that vitamin D activities involve vitamin D receptor (VDR)-dependent and VDR-independent effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 25-hydroxyvitamin D3 (25(OH)D3) and ligand-independent effects of the VDR. Here, we describe a novel in vivo system using genetically modified rats deficient in the Cyp27b1 or Vdr genes. Type II rickets model rats with a mutant Vdr (R270L), which recognizes 1,25(OH)2D3 with an affinity equivalent to that for 25(OH)D3, were also generated. Although Cyp27b1-knockout (KO), Vdr-KO, and Vdr (R270L) rats each showed rickets symptoms, including abnormal bone formation, they were significantly different from each other. Administration of 25(OH)D3 reversed rickets symptoms in Cyp27b1-KO and Vdr (R270L) rats. Interestingly, 1,25(OH)2D3 was synthesized in Cyp27b1-KO rats, probably by Cyp27a1. In contrast, the effects of 25(OH)D3 on Vdr (R270L) rats strongly suggested a direct action of 25(OH)D3 via VDR-genomic pathways. These results convincingly suggest the usefulness of our in vivo system.

Generation of 1,25-dihydroxyvitamin D3 in Cyp27b1 knockout mice by treatment with 25-hydroxyvitamin D3 rescued their rachitic phenotypes.

We have reported that 25-hydroxyvitamin D3 [25(OH)D3] binds to vitamin D receptor and exhibits several biological functions directly in vitro. To evaluate the direct effect of 25(OH)D3 in vivo, we used Cyp27b1 knockout (KO) mice, which had no detectable plasma 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] when fed a diet containing normal Ca and vitamin D. Daily treatment with 25(OH)D3 at 250 µg kg-1 day-1 rescued rachitic phenotypes in the Cyp27b1 KO mice. Bone mineral density, female sexual cycles, and plasma levels of Ca, P, and PTH were all normalized following 25(OH)D3 administration. An elevated Cyp24a1 mRNA expression was observed in the kidneys, and plasma concentrations of Cyp24a1-dependent metabolites of 25(OH)D3 were increased. To our surprise, 1,25(OH)2D3 was detected at a normal level in the plasma of Cyp27b1 KO mice. The F1 to F4 generations of Cyp27b1 KO mice fed 25(OH)D3 showed normal growth, normal plasma levels of Ca, P, and parathyroid hormone, and normal bone mineral density. The curative effect of 25(OH)D3 was considered to depend on the de novo synthesis of 1,25(OH)2D3 in the Cyp27b1 KO mice. This suggests that another enzyme than Cyp27b1 is present for the 1,25(OH)2D3 synthesis. Interestingly, the liver mitochondrial fraction prepared from Cyp27b1 KO mice converted 25(OH)D3 to 1,25(OH)2D3. The most probable candidate is Cyp27a1. Our findings suggest that 25(OH)D3 may be useful for the treatment and prevention of osteoporosis for patients with chronic kidney disease.

[A case of Cronkhite-Canada syndrome in which contrast radiologic examinations of the digestive tract were performed at the time of development of the clinical symptoms].

We encountered a case of Cronkhite-Canada syndrome in which contrast radiologic examinations of the upper and lower digestive tract were performed immediately before and after the development of the clinical symptoms. These contrast radiologic images showed mainly mucosal coarseness and no polyposis of the stomach and colon. The endoscopy, performed 3 months later from the development of the clinical symptoms, revealed polyposis of the stomach and colon. So we recognized that the clinical symptoms developed before appearance of the polyposis of the digestive tract and the polyposis progressed rapidly.

[A case of female alcoholic who developed liver cirrhosis with small amounts of alcohol by the common use of contraceptive agent].

A case of female alcoholic who developed liver cirrhosis with small amounts of alcohol by the common use of contraceptive agent was reported. A case was a 33-year-old female who had complained of systemic edema and jaundice. She had been drinking alcohol, while she had been taking the contraceptive agent from 20-year-old. On admission, she had a large amount of ascites with jaundice. She was diagnosed as liver cirrhosis and hepatic failure by CT scanning of abdomen and laboratory data. Her condition was temporarily improved by the abstinence and the treatment. Since she drank under hospitalization, she had to change the hospital and died after 2 months. She had been drinking for only 10 years. Her cumulative alcohol intake was also very small. She may have developed alcoholic cirrhosis with small amount of alcohol because of common use of contraceptive agent with drinking.

Overlap/switch to adefovir monotherapy for lamivudine-resistant patients who responded to combination therapy: a pilot controlled study.

OBJECTIVE: The aim of this study was to investigate the outcome of overlap/switch to adefovir dipivoxil (ADV) monotherapy for chronic hepatitis B (CHB) patients with lamivudine (LAM)-resistant HBV, who responded to LAM plus ADV combination therapy. METHODS: In 29 of 35 LAM-resistant CHB patients, serum HBV-DNA levels decreased to <3.7 log genome equivalent (LGE)/mL at 12 months after LAM plus ADV combination therapy, defined as complete virological response (CVR). The 29 CVR patients were randomly allocated to continuation of combination therapy or switch to ADV monotherapy within 12 months. The cumulative rates of sustained CVR were compared between the two groups. RESULTS: The follow-up duration after randomization was 19.3-36.7 months (median, 28.2 months) for the combination group and 21.0-36.4 months (29.0 months) for the overlap/switch group. The cumulative rate of sustained CVR during the follow-up period was 100% in all patients of both groups. The total medical expenses during follow-up after randomization were median US$20,949 for the combination group and US$16,107 for the overlap/switch group (p=0.012). Overlap/switch to ADV monotherapy sufficiently repressed the replication of LAM-resistant mutants without the development of ADV-resistant mutants. The rate of sustained CVR was not influenced by treatment regimen (continuation of combination therapy or switching to ADV monotherapy), the duration of the overlap period, or patient and virological characteristics. CONCLUSION: In LAM-resistant CHB patients who achieved CVR to LAM plus ADV combination therapy, CVR was maintained after overlap/switch to ADV monotherapy, suggesting that it could be a useful regimen for such patients.

Experimental studies on the relationship between immune responses and liver damage induced by ethanol after immunization with homologous acetaldehyde adducts.

BACKGROUND: It has been considered that acetaldehyde (AcH) adducts induce liver injury through an immune response. Previous experimental studies showed that hepatic necrosis, inflammatory cell infiltration, and hepatic fibrosis were induced in guinea pigs immunized with heterologous human AcH adducts and ethanol feeding. AcH modification of foreign protein may markedly increase immunogenicity of the protein itself, leading to enhanced formation of immune complex and possible liver injury. The present study investigated whether immune responses and alcoholic liver disease would be induced in mice by immunization with mouse albumin-AcH adducts and ethanol feeding. METHODS: 6B6 mice were divided into six groups with or without immunization and ethanol feeding. Mice were immunized with mouse albumin-AcH adducts three times at 2-week intervals and fed ethanol for 10 weeks. The stimulation index of [(3)H]thymidine uptake into lymphocytes cultured with mouse albumin or mouse albumin-AcH adducts was measured. Histologic findings of the liver were examined, and the plasma levels of aspartate transaminase and alanine aminotransferase were also measured. RESULTS: The stimulation index was increased remarkably in ethanol-fed mice that were immunized with mouse albumin-AcH adducts. However, neither inflammatory cell infiltration nor hepatic necrosis was observed in the liver. There were also no differences in the plasma activities of aspartate transaminase and alanine aminotransferase between the group of mice regardless of ethanol feeding or immunization. CONCLUSION: Although marked immune responses were observed, no liver damage was induced by long-term ethanol feeding in our mouse model using AcH-homologous albumin adducts. These results suggest that homologous protein adducts may not induce liver injury by long-term ethanol feeding or may have lower immunogenicity than heterologous protein adducts. These results also suggest that nonreduced AcH adducts and/or a larger amount of ethanol may be needed for liver injury in this model.