Cell surface adenosine 5'-triphosphatase as an in vitro marker of the lineage and cytodifferentiation of oncogenic epithelial cells from rat liver parenchyma.

The relationship between the oncogenicity and the surface properties of cultured liver epithelial cells has been studied with the newborn Wistar rat-derived euploid line, RL34, and its heteroploid variants. An oncogenic variant, RL34HT, appeared to be more functionally active than its nononcogenic counterparts with respect to cell surface adenosine 5'-triphosphatase (ecto-ATPase) as well as to cytoplasmic enzymes such as tyrosine aminotransferase, gamma-glutamyl transpeptidase, and alkaline phosphatase. The cell surface of RL34HT was distinguished from those of nononcogenic and marginally oncogenic cell populations by the presence of abundant microvilli and by the absence of large external transformation-sensitive protein (fibronectin). High-Km and high-Vmax Ca2+-Mg2+ -ecto-ATPase was found in RL34HT. All nononcogenic cell lines had a flat granular surface membrane with high levels of fibronectin and also exhibited ecto-ATPase activity with low Km and low Vmax. When RL34HT was grown in dibutyryl cyclic adenosine 3',5'-monophosphate and theophylline, the external cell surface was partially restored to the polypeptide compositions of RL34, and there was an increase in Vmax of ecto-ATPase without a change in Km. The high-Km ecto-ATPase may be a useful indicator reflecting the lineage and cytodifferentiation of oncogenic liver epithelial cells, since it is also known to be localized at the bile canalicular microvilli of normal adult hepatocytes.

The anticancer effect of chaetocin is enhanced by inhibition of autophagy.

Chaetocin is a fungal metabolite that possesses a potent antiproliferative activity in solid tumors by inducing cell death. Although recent studies have extended the role of chaetocin in tumors, the underlying molecular mechanisms such as the downstream cascade that induces cell death has not clearly been elucidated. In this study, we show that chaetocin is able to induce both apoptosis and autophagy in several hepatoma cell lines including HepG2, Hep3B and Huh7 cell lines. Moreover, we found that the inhibition of caspase-3/7 activity by z-VAD-fmk treatment was able to block chaetocin-mediated cell death, whereas blocking autophagy by Bafilomycin A1 or the knockdown of autophagy protein 5 enhanced cell death mediated by chaetocin. These findings suggest that chaetocin has a potent anticancer effect against hepatoma. Inhibition of autophagy may potentiate anticancer effects of chaetocin thus providing evidence that combined treatment with chaetocin and autophagy inhibitors will be an effective strategy for treating cancer.

p21(WAF1) is associated with CDK2 and CDK4 protein during HL-60 cell differentiation by TPA treatment.

TPA-treated HL-60 cells are mainly arrested in G1 by p21(WAF1) accumulation. We investigate the downstream changes following such accumulation. Increased p21(WAF1) is associated with CDK2 and CDK4. pRb is dephosphorylated in the presence of p21-CDK2/4 complexes, and the Rb-E2F1 complex increases after TPA treatment, whereas the Rb-HDAC1 complex decreases slightly. Our results suggest that increased p21(WAF1) is associated with CDK2/4, and that these complexes induce pRb dephosphorylation. In turn, hypophosphorylated pRb are mainly complexed with E2F1, but HDAC1 appears not to be a key component in this process.

Immune response induced by immunization with Hepatitis B virus core DNA isolated from chronic active hepatitis patients.

There are many mutations in the gene encoding Hepatitis B virus (HBV) core antigen of chronic active hepatitis patients, and such mutations are most likely to be related to the severity of disease. Here, we constructed plasmids containing wild-type and deletion type of HBV core gene (HBc) to develop an experimental DNA vaccine and to compare immunogenicity of two types of HBc vaccine. Twenty-nine wild-types and seven deletion types of HBc were detected in sera of 32 Korean patients with chronic active hepatitis. Four wild-types (W1, W2, W4, W6) and two deletion types (D3, D4) of HBc were cloned into the pcDNA3 vector. Intramuscular immunization with wild-type HBc efficiently increased serum anti-HBc antibody response in a dose-dependent manner. Anti-HBc antibody response in mice injected with W6 increased 14 days after immunization, and peaked after 30 days and was maintained at least up to 50 days. W6 immunization induced a specific cytotoxic T lymphocyte response to W6-transfected 3LL (3LL-W6), and reduced the sizes of tumor mass of mice challenged with 3LL-W6 or 3LL transfected with D4. However, intramuscular immunization with D3 and D4 did not show antibody response at all. D3 and D4 have 157 bp (from 331 to 491 bp) and 122 bp (from 327 to 448 bp) gene deletion, respectively, and these encode class II MHC-restricted T-cell epitope. Altogether, these results suggest that mutant virus that has deleted HBc gene may evade immune systems due to loss of T-cell epitope.

Lack of mutation at p16INK4A gene but expression of aberrant p16INK4A RNA transcripts in human ovarian carcinoma.

Alterations of the p16INK4A gene are frequent in various human cancers. We investigated p16INK4A gene status in 20 ovarian carcinomas by PCR (polymerase chain reaction), PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and sequencing techniques. None of the primary tumors showed any mutational or deletional events. However, 19 out of 20 tumors displayed both a methylated and an unmethylated p16INK4A promoter. In some of these samples, we detected aberrant p16INK4A transcripts, with partial deletions of both exons 1 and 2, which could not encode a functional p16INK4A protein. The sequences of the aberrant mRNA revealed common 4-7 nucleotide sequences before and after the deleted region, which might cause abnormal splicing of mRNA transcripts. These results suggest that both promoter methylation and aberrant mRNA processing may interfere with p16INK4A expression in ovarian tumors.

5-Aza-2'-deoxycytidine leads to down-regulation of aberrant p16INK4A RNA transcripts and restores the functional retinoblastoma protein pathway in hepatocellular carcinoma cell lines.

The inactivation of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor p16INK4A may be caused by gene deletion, mutation or promoter hypermethylation. We have previously reported that p16INK4A in hepatocellular carcinoma (HCC) tissues and cell lines is inactivated predominantly by promoter hypermethylation rather than genomic aberrations. In the present experiments, we have studied the effects of the demethylating agent, 5-aza-2'-deoxycytidine (5-AZA/decitabine), on the expression of aberrant p16INK4A RNA transcripts and the CDK-retinoblastoma gene pathway in HCC cell lines with p16INK4A promoter hypermethylation. The expression of aberrant p16INK4A RNA transcripts was down-regulated and p16INK4A protein was strongly re-expressed in the HCC cell lines, SNU 354, 398, 423 and 475 after 5-AZA/decitabine treatment for 5 days. The re-expressed p16INK4A was functional, because it bound to and inhibited CDK4 kinase activity, and increased the concentrations of the hypophosphorylated form of retinoblastoma protein (pRB) in cells with a wild type RB gene. Moreover, treatment with the demethylating agent led not only to G1 cell cycle arrest, but also to the increased expression of the senescence-associated marker beta-galactosidase. This up-regulation of p16INK4A mRNA and protein correlated with demethylation of the p16INK4A promoter, and with the down-regulation or disappearance of aberrant p16INK4A transcripts. These results suggest that the aberrant p16INK4A RNA transcript can be transcribed from the methylated p16INK4A gene, and endogenous reactivation of functional p16INK4A mRNA by a demethylating agent can restore the pRB pathway in HCC, and foster the terminal differentiation of the malignant cells. Therefore, demethylating agents, such as 5-AZA/decitabine, may have potential in the treatment of HCC.

Chemopreventive agent resveratrol, a natural product derived from grapes, reversibly inhibits progression through S and G2 phases of the cell cycle in U937 cells.

Resveratrol, a natural product derived from grapes, has been shown to prevent carcinogenesis in murine models. We report here that resveratrol induces antiproliferation and arrests the S phase in human histiocytic lymphoma U937 cells. Resveratrol induces arrest in the S phase at low concentrations (30-60 microM), but high concentrations do not induce S phase accumulation in U937 cells. Removal of resveratrol from the culture medium stimulates U937 cells to reenter the cell cycle synchronously, as judged by the expression patterns of cyclin E, A and by fluorescent activated cell sorting analysis. These data demonstrate that resveratrol causes S phase arrest and reversible cell cycle arrest. Thus, resveratrol provides an important new cell cycle blocker as well as a cancer chemopreventive agent.

Collagen mRNA expression detected by in situ hybridization in keloid tissue.

The keloid fibroblasts exhibited increased extracellular matrix gene expression, and prominent elevated type I procollagen mRNA when compared to control fibroblasts cultured from the uninvolved skin of normal people. It also showed markedly elevated type I/III procollagen mRNA ratios, but no synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. By in situ hybridization in keloid tissue, high levels of type I and type III procollagen mRNAs were detected in most of the fibroblasts, suggesting the presence of a subpopulation responsible for the increased collagen production. The levels of type I and type III procollagen mRNAs in these fibroblasts were clearly elevated compared to control skin specimens. And concentration of type I procollagen mRNA was found more predominantly than was type III. These results suggest that deposition of collagen in keloid could result from activation of certain fibroblasts responsible for type I procollagen production.

The green tea polyphenol (-)-epigallocatechin gallate attenuates beta-amyloid-induced neurotoxicity in cultured hippocampal neurons.

Previous evidence has indicated that the neuronal toxicity of amyloid beta (betaA) protein is mediated through oxygen free radicals and can be attenuated by antioxidants and free radical scavengers. Recent studies have shown that green tea polyphenols reduced free radical-induced lipid peroxidation. The purpose of this study was to investigate whether (-)-epigallocatechin gallate (EGCG) would prevent or reduce the death of cultured hippocampal neuronal cells exposed to betaA because EGCG has a potent antioxidant property as a green tea polyphenol. Following exposure of the hippocampal neuronal cells to betaA for 48 hours, a marked hippocampal neuronal injuries and increases in malondialdehyde (MDA) level and caspase activity were observed. Co-treatment of cells with EGCG to betaA exposure elevated the cell survival and decreased the levels of MDA and caspase activity. Proapoptotic (p53 and Bax), Bcl-XL and cyclooxygenase (COX) proteins have been implicated in betaA-induced neuronal death. However, in this study the protective effects of EGCG seem to be independent of the regulation of p53, Bax, Bcl-XL and COX proteins. Taken together, the results suggest that EGCG has protective effects against betaA-induced neuronal apoptosis through scavenging reactive oxygen species, which may be beneficial for the prevention of Alzheimer's disease.

Patterns of progression of localized retinal nerve fibre layer defect on red-free fundus photographs in normal-tension glaucoma.

PURPOSES: To investigate patterns of progression of localized retinal nerve fibre layer (RNFL) defect on red-free fundus photographs and to quantify extents of progression in normal-tension glaucoma (NTG) patients. METHODS: Sixty-five eyes of consecutive 65 NTG patients who had shown progression of localized RNFL defect on serial red-free fundus photographs were selected for this study. Patterns of progression of localized RNFL defect on red-free fundus photographs were categorized and extents of progression were quantified. Serial assessments of disc stereophotographs and visual fields were also performed to detect progression. RESULTS: The most common pattern of progression was widening of the defect towards the macula (n=37; 56.9%) followed by deepening of the defect (n=25; 38.5%), appearance of a new defect (n=6; 9.2%), and widening of the defect away from the macula (n=5; 7.7%). Eight eyes simultaneously showed two patterns of progression. Mean angular widening of the defect towards the macula and away from the macula was 6.4+/-4.1 degrees (range: 1.1-17.1 degrees , n=37) and 3.4+/-2.1 degrees (range: 1.1-5.2 degrees , n=5), respectively. No progression was observed on the disc stereophotographs (n=65) or in the visual fields (n=55) in 64 eyes (98.5%) and 46 eyes (83.6%), respectively. CONCLUSIONS: There were four patterns of progression of localized RNFL defect. In most cases, RNFL loss proceeded temporally.

[A study on the influentials of knowledge gap].

PIP: This study investigates the influence of structural and functional characteristics on the knowledge gap of family planning and sex information in unmarried female workers. In explaining the knoledge gap, structural variables, such as age, education, and economic status, as well as functional variables, such as salience, the are regarded as independent variables, while seeking exposure to related information is regarded as a mediating variable. Using 1983 data collected by the Korea Instituted for Population and Health for a study on knowledge and attitudes on population and sex among unmarried female workers, the topics of family planning and sex informaton are discussed. Results show that age, education, salience, and seeking exposure have significant effects on the difference of group means on family planning and sex knowledge; only economic variables show no relations at all. A multiple regression analysis, using a path model, shows that education and salience explain 32% of the variance in seeking exposure. In explaining reproductive physiological knowledge, exposure explains 29% of the variance, while education explains 4%. Salience and age do not explain anything. In contraceptive knowledge seeking exposure explains 26% of the variance, followed by salience at 5%. The knowledge gap on physiology forms structurally, while the knowledge gap on contraceptive use forms functionally. Overall, education and salience determine seeking exposure level, and this affects the knowledge level. Therefore, family planning and sex education through schools or institutional education must be strengthened; before the unmarried female worker becomes too involved in the opposite sex, enough information to have a healthy social life must be learned.^ieng

[A study on characteristics of active family planning canvassers (author's transl)].

PIP: The importance of active canvassers as voluntary family planning manpower has been well recognized. The purpose of this study was to determine the sociodemographic characteristics, family planning message exposure, and knowledge, attitude, and practice of these active voluntary canvassers using 1978 national survey data. We operationally defined the active canvassers using 2 kinds of questions: 1) Have you ever canvassed family planning to young couples? and 2) Have you ever canvassed contraception to a neighborhood? If a woman has canvassed both, she was considered an active canvasser. And if a woman had no canvass experience, she was considered 1 of the inactive group. In this analysis, the less actives who had canvassed either of the 2 were omitted. The active canvassers seemed to have distinct sociodemographic characteristics as compared with the inactive. Those aged 35-9 with more than 2 children (at least 1 son) tended to be active canvassers. Those who lived in urban areas, who had more than 6 years of education, a relatively high living standard, and who participated in economic activities also showed strong tendencies towards canvassing. The active canvassers are exposed to relatively more family planning messages through various media. Their personal source had significant impact on canvas activities regardless of residence or educational level. Family planning message exposure through mass media also showed significant relationship with canvass activities except those who lived in rural areas and who had more than 6 years of education. The active canvassers also tended to have more specific knowledge about each method and they also showed a more favorable attitude toward family planning and had a higher practice rate than the inactives. But when we consider such demographic characteristics as age or number of living sons of the active canvassers, there is a possibility that women do not participate in canvassing until they have achieved their desired family size (including number of sons). (Author's modified)^ieng

The effect of reproductive intentions on subsequent fertility among low-parity Korean women, 1971-76.

The effect of reproductive intentions on subsequent fertility is evaluated for a group of low-parity, primarily rural, Korean women. These women were more successful in achieving wanted additional births than in avoiding unwanted births, both at the aggregate and individual level. Several models of fertility behavior are tested. Reproductive intentions and number of living sons are the best predictors of subsequent fertility, and their effects are mediated largely by their impact on induced abortion. Overall the effect of reproductive intentions on subsequent fertility among Korean women is very similar to findings reported for the United States and Taiwan.

PIP: The effect of reproductive intentions on subsequent fertility is evaluated for a group of low parity, primarily rural, Korean women. These women were more successful in achieving wanted additional births than in avoiding unwanted births, both at aggregate and individual levels. Several models of fertility behavior are tested. Reproductive intentions and number of living sons are the best predictors of subsequent fertility, and their effects are mediated largely by their impact on induced abortion. Overall, the effect of reproductive intentions on subsequent fertility among Korean women is very similar to findings reported for the U.S. and Taiwan.

[Analysis of contraceptive use: 1968-1991].

PIP: The contraceptive use rate in 1991 was 79.4%, which showed that the contraceptive use rate had reached the saturation point in Korea. In fact, the rapid increase in the use rate was evaluated as one of three major contributors in decreasing the TFR below the replacement level along with marriage age and induced abortion. The objective of this study was to analyze the determinants of contraceptive use by method in 1968, 1974, 1982, 1988, and 1991 using logistic regression as the statistical method. The results showed a significant relationship between sociodemographic variables and contraceptive use. The effect was strongest in 1968. Even though the relationship was somewhat weaker in 1988, it was strong again in 1991. This result contradicts the idea that there may only be a weak or no relationship between socioeconomic variables and contraceptive use owing to the effect of popular contraceptive use and increased availability of contraceptives. The relatively weak relationship in 1988 can be explained by the reinforced family planning programs of the government during the 1980s. Based on the analysis, it maybe learned that in spite of the high contraceptive use rate part of the population is still subject to unwanted pregnancies; so the government should endeavor to provide qualitative services to improve the quality of life as well as maintain an appropriate fertility level.^ieng

[Area differentials of sex imbalance in births].

Along with a rapid decline in fertility to below replacement levels in South Korea, a new demographic phenomenon has emerged: a distorted sex ratio at birth. In 1993, the sex ratio was 115.6, with significant differences in this ratio by region. "An analysis of national survey data showed that in the areas with the most distorted imbalance, people not only have a stronger preference for sons and more favoring attitude toward sex-selective induced abortion, but also actually have more sex-selective induced abortions after checking the sex of the fetus. This analysis proved that the sex imbalance is being caused by means of sex-selective induced abortions after checking the sex of the fetus." (SUMMARY IN ENG)

Isolation and characterization of nifDK::kanamycin and nitrogen fixation proficient Azotobacter vinelandii strain, and its implication on the status of multiple chromosomes in Azotobacter.

Several lines of experimental analyses on the ploidy status of Azotobacter vinelandii genome lead to the conclusion that it contains more than 40 copies of its chromosome and therefore it is a polyploid organism. The genetic evidence argues against the existence of polyploidy in these cells since the segregation pattern of genetic markers under lack of selection pressure mimic that of haploids. However, when A. vinelandii was made Nif- by inserting a kanamycin resistance marker gene in the nifDK sequence and the cells were selected for kanamycin resistance and Nif+ phenotype, we were able to score colonies that are both kanamycin resistant and Nif+. Therefore, when the cells were subjected to forced double selection of the same locus, they behaved as if they carried at least two chromosomes, one carrying the kanamycin resistance marker in the nifDK genes and the other carrying the intact nifDK genes. These analyses suggested that at least a diploidy status can be induced in these cells under selection pressure.

Impact of the national family planning program on fertility in rural Korea: a multivariate areal analysis.

The impact of the previous three years' input of the national family planning program on 1975 fertility levels in rural Korea was assessed using multivariate areal analysis techniques. Program input had a measurable negative effect on fertility, but the magnitude of the effect was lower than that of nonprogram factors. Holding availability of nonprogram methods of fertility limitation constant strengthened the impact of program input. It was suggested that the effect of the national program was statistically obscured by the selective concentration of program input in high-fertility, physician-poor areas.

PIP: The impact of the previous 3 years' input of the national family planning program on 1975 fertility levels in rural Korea was assessed using multivariate and areal analysis techniques. Program input had a measurable negative effect on fertility, but the magnitude of the effect was lower than that of nonprogram factors. Holding availability of nonprogram methods of fertility limitation constant strengthened the impact of program input. It was suggested that the effect of the national program was statistically obscured by the selective concentration of program input in high fertility, physician-poor areas.

R plasmids conferring multiple drug resistance from shigella isolated in Korea.

The majority (85%) of shigella isolated in 1980 and 1981 in Korea were Shigella flexneri, the others were Sh. sonnei (14%) with only a small number of Sh. dysenteriae. Only 14 of the 459 strains of shigella isolated were susceptible to all 12 drugs tested, and 445 were resistant to three or more drugs. Strains multiply resistant to the six drugs, chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm), sulfisomidine (Su), ampicillin (Ap) and trimethoprim (Tp) were most frequently encountered, followed by those resistant to Cm, Tc, Sm, Su and Tp. The complete patterns of resistance to drugs except nalidixic acid and rifampin in approximately 73% of drug-resistant strains were co-transferred to Escherichia coli by conjugation, indicating that the resistance was R plasmid-mediated. Randomly selected R plasmids conferring various patterns of resistance markers were tested for the incompatibility groups, and almost all of them were classified into Inc FII. Two of three R plasmids conferring resistance to Cm, Tc, Sm and Su were classified into Inc B and one to Inc FII. Two R types with resistance markers of Cm, Tc, Sm and Ap were not classified with our standard plasmids used.

Persistent expression of Fas/FasL mRNA in the mouse hippocampus after a single NMDA injection.

Synaptic reorganization plays a very important role in brain adaptations to environmental stimuli, diseases, and aging processes. The NMDA model of excitotoxic injury was used to investigate the long-term molecular changes in the surviving neural cells in the mouse hippocampus. We demonstrated that a single intraperitoneal injection of NMDA produces persistent expression of c-fos, c-jun, Fas, and Fas ligand (FasL) mRNA in the hippocampus for 5 months. To determine the cellular origin of those gene transcripts in our in vivo model, a glial cell line and primary fetal neuronal culture were used to investigate the inducibility of the c-fos, c-jun, Fas, and FasL mRNA by NMDA. Both c-fos and Fas mRNA expression was observed in the NMDA-treated glial or neuronal cultures; however, c-jun and FasL mRNA was undetectable in this study. In our in vivo model, mossy fiber sprouting and apoptosis were also observed up to 40 days after the NMDA injection. Therefore, we hypothesize that the observed long-term expression of c-fos, c-jun, Fas, and FasL mRNAs may reflect the ongoing synaptic reorganization.

DNA-mediated immunization of mice with plasmid encoding HBs antigen.

In order to develop an experimental DNA vaccine for the prevention and treatment of hepatitis B virus infection, hepatitis B virus surface antigen (HBsAg) DNA was subcloned into an E. coli-eukaryotic cell shuttle vector and was expressed in the Baculovirus expression system. Intramuscular, intradermal, and intraperitoneal injections of 30 microg of the plasmid DNA expressing HBsAg induced humoral and cellular immune responses in ICR mice. The first IgG antibodies were detected after ten days and specific IgG antibody titers peaked after two months of a single intramuscular DNA injection. Anti-HBs antibody titers gradually increased and peaked at four months following intradermal DNA injection, and in case of intraperitoneal injection they peaked at seven months. Generation of HBs-specific helper T lymphocytes was also investigated through the production of interleukin-2 by T helper cells. Boosting effects of HBs DNA were investigated without much results. In general, DNA-mediated HBs immunization induced humoral and cellular immune responses in mice that appears to simulate immune responses in human during the course of HBV vaccination.