Enhancement in the cleavage activity of a hammerhead ribozyme by cationic comb-type polymers and an RNA helicase in vitro

The activity of a hammerhead ribozyme (Rz) in vivo depends on several factors, such as abundance, stability, and accessibility of Rz to its target mRNA. Among these factors, accessibility is believed to be the rate-limiting factor for Rz-mediated cleavage in vivo. As Rz and its substrate RNA are negatively charged, we examined whether cellular RNA-interacting proteins or artificial polycations might improve the accessibility of Rz to its substrate RNA. Specifically, we examined the effects of two kinds of cationic comb-type copolymer, alphaPLL-g-Dex, and a cellular RNA helicase on the accessibility of Rz to a model structured RNA in vitro. The cleavage activity of Rz was slightly enhanced by alphaPLL-g-Dex, probably due to an acceleration of the association/dissociation rate. And also, the RNA helicase-bound hybrid-Rz could cleave the target substrate at a significantly higher rate due to its unwinding activity for the duplex RNA substrate. These approaches should be useful in the development of efficient gene-inactivating reagents in the post-genomic era.     

Long-term Helicobacter pylori infection in Japanese monkeys induces atrophic gastritis and accumulation of mutations in the p53 tumor suppressor gene

Background. Helicobacter pylori is accepted as a definite human gastric carcinogen from an epidemiological point of view despite insufficient experimental data. Although we previously showed that the number of p53 immunopositive cells in the atrophic gastric mucosa of H. pylori‐infected Japanese monkeys gradually increased over time, data on p53 gene mutations were not obtained in that study. To obtain direct evidence of carcinogenesis associated with H. pylori infection, we investigated whether p53 gene mutations are present in the gastric mucosa of a nonhuman primate model susceptible to H. pylori. Materials and Methods. Using the DNA from gastric tissues obtained from six H. pylori‐uninfected monkeys of different ages, nucleotide sequence of the wild‐type p53 gene was determined by amplification of exons (Ex) 5, 6, 7 and 8 and sequencing. Gastric specimens obtained from eight Japanese monkeys that had been infected with H. pylori for different lengths of time (1.5–7.5 years), were analyzed for mutations in exons 5–8 of p53. Results. In the six H. pylori‐uninfected monkeys, nucleotide sequences of p53 Ex 5–8 were completely common and no mutations were noted. However, among the monkeys that were infected with H. pylori over various periods of time, there was an accumulation of p53 nucleotide (amino acid) substitutions as the gastric atrophy score increased. Conclusions. We conclude that the appearance of p53 gene mutation may be closely associated with the degree of gastric mucosal atrophy, which depends on the duration of H. pylori infection. Searching for p53 gene mutations may be useful for studying the progression of gastric carcinogenesis associated with H. pylori.     

Enterocromaffin-like cell tumor induced by Helicobacter pylori infection in Mongolian gerbils

Background. Gastric carcinoids are strongly associated with chronic atrophic gastritis A, and it is suggested that hypergastrinemia plays a critical role in development of gastric carcinoids. Since Helicobacter pylori infection causes hypergastrinemia, it is held that H. pylori infection produces gastric carcinoids. We followed the histological changes of H. pylori‐infected stomachs of Mongolian gerbils for a long time. Materials and Methods. Five‐week‐old‐male Mongolian gerbils were infected with H. pylori ATCC 43504 with cagA gene, expressing vacuolating cytotoxin. Determination of the serum gastrin and histopathological examination of the stomach at 6, 12, 18, and 24 months after H. pylori inoculation was studied and compared with uninfected animals . Results In infected animals, the gastric carcinomas appeared 18 and 24 months after infection. Endocrine cell dysplasias and carcinoids with marked atrophic gastritis of the oxyntic mucosa were observed in the infected animals 24 months after H. pylori inoculation. The serum gastrin level in the infected group increased from an average of 86.2 pg/ml at the beginning of the study to an average of 498 pg/ml and 989 pg/ml at 18 and 24 months after infection, respectively. These changes in the serum gastrin levels were significant compared with uninfected controls that showed no changes. Conclusions. H. pylori infection caused not only gastric carcinomas but also enterochromaffin‐like cell tumors in Mongolian gerbils, due to hypergastrinemia. This model is thought to be useful to study the relationship between hypergastrinemia and gastric carcinoids.     

A fast, simple method for screening radiation susceptibility genes by RNA interference

Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes.     

Changes in the morphology of cell-size liposomes in the presence of cholesterol: formation of neuron-like tubes and liposome networks

Spontaneous changes in the morphology of cell-size liposomes (dioleoylphosphatidylcholine, DOPC and egg PC) as model cells were investigated in the presence of cholesterol. Tube structures and liposome networks connected by the tubes were observed in the presence of 5-30% cholesterol by dark-field and laser-scanning microscopy. Furthermore, in the presence of more than 40 mol% of cholesterol, the tubes disappeared and changed to small liposomes. Thus, cholesterol induced a morphological change in giant liposomes from tubes to small liposomes. These phenomena may be related to the role of cholesterol in the morphological changes in living cells such as neurons.     

Establishment of stable hFis1 knockdown cells with an siRNA expression vector

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.     

Activation of the CKI-CDK-Rb-E2F pathway in full genome hepatitis C virus-expressing cells

Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.     

Use of a randomized hybrid ribozyme library for identification of genes involved in muscle differentiation

We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.     

Augmenting effect of antibiotics on endotoxin activity may cause a safety problem

Enhancing/interfering effect of antibiotics on endotoxin was evaluated using the endotoxin test and the cell line assay in 28SC cells that has a responsiveness consistent with that of human peripheral blood. When a total of 21 products of seven different kinds of antibiotics were tested, none showed any significant effect on the endotoxin test at its therapeutic dose. However, aminoglycosides showed a significant augmenting effect on IL-6 induction of endotoxin in 28SC cells. Detailed examination of the augmenting effect was made on spectinomycin in the in vitro cell line assay and also in the lethal endotoxin challenge assay in D -galactosamine-treated mice. Spectinomycin also enhanced the endotoxin lethality in D -galactosamine-treated mice. A kinetic analysis in endotoxin-sensitized 28SC cells revealed that the augmentation takes place as quickly as 10 min after spectinomycin treatment. Accordingly, a special caution concerning the augmenting effect was assumed necessary for the safety control of antibiotic products as well as for selecting antibiotics for the therapeutic use.