Awareness Survey on Community Water Fluoridation by Region / 치위생과학회지

Background@#The purpose of this study is to investigate the pro-con of re-implementation by administrative areas and the difference in perception of community water fluoridation in implemented and non-implemented areas after the community water fluoridation in Korea was suspended. Through this, we intend to provide basic data that can help find ways to increase the support and interest of local residents. @*Methods@#The 601 questionnaires collected through the survey and statistical analysis was conducted using SPSS Statistics 28.0. @*Results@#As a result of analyzing the perception of the community water fluoridation according to the understanding of fluorine, the proportion of people who were not recognized by both fluorine and community water fluoridation was the highest (p＜0.05). As a result of the analysis of the pro-con of re-implementation of community water fluoridation, the approval was high. Among those who responded in favor, the place of re-implementation showed that ‘implementation nationwide’ was high. As for the reason for favor, it was found that it was possible to prevent dental caries disease. The reason for the objection was the lack of knowledge about fluoride. @*Conclusion@#The results of the survey for the pro-con of the re-implementation of community water fluoridation showed a higher degree of ‘agree’ and showed that people in the area where community water fluoridation was not implemented showed higher interest in oral health prevention and management. Through this, not only oral education, but also correct information on the implementation method of community water fluoridation, the benefits of community water fluoridation, and the facts that were misunderstood in the past, as well as oral education, can be provided to raise interest in community water fluoridation. It is thought that the expected effect of the re-implementation of community water fluoridation can be obtained if such activities are carried out.

Study on the characterization, biocompatibility and efficacy of sponge brush products for oral care / 대한치과재료학회지

A sponge brush is an oral hygiene product that is used to clean buccal, gingival and palatal mucosa. The aim of this study was to observe the appearance, porosity, water sorption, cytotoxicity and efficacy of sponge brush products. Seven commercially available sponge brush products were used as test materials. To observe the appearance of sponge brush, the dimension and weight were examined. The porosity was measured using the micro CT. The water sorption rate was calculated by weight change before and after immersion of sponge brush in distilled water for 5 s. The sponge brush was extracted according to the KS P ISO 10993-12, and examined the extracted solution by visual inspection. Extracted solution was then evaluated with MTT assay. Protein removal efficacy of sponge brush product from the bovine serum albumin coated sheet was tested using bicinchoninic acid assay. The results of the length and diameter range of brush were 17~24 mm and 14~24 mm respectively.The length and thickness range of the handle was 14~16 cm and 3~5 mm respectively. There were significant differences in the sponge brush weight of all products (p<0.05), and porosity of the sponge was indicated to be more than 80% in all products.The water sorption rate ranged from 21 to 62% (p<0.05). As the results of extraction, there were neither foreign materials nor impurities in all of sponge brush products. The cell viability was indicated to be more than 70% except for two products.After applying all sponge brush products, the protein removal rate was more than 70%. The above results will provide useful information for future development of related oral care products.

Study on the characterization, biocompatibility and efficacy of sponge brush products for oral care / 대한치과재료학회지

A sponge brush is an oral hygiene product that is used to clean buccal, gingival and palatal mucosa. The aim of this study was to observe the appearance, porosity, water sorption, cytotoxicity and efficacy of sponge brush products. Seven commercially available sponge brush products were used as test materials. To observe the appearance of sponge brush, the dimension and weight were examined. The porosity was measured using the micro CT. The water sorption rate was calculated by weight change before and after immersion of sponge brush in distilled water for 5 s. The sponge brush was extracted according to the KS P ISO 10993-12, and examined the extracted solution by visual inspection. Extracted solution was then evaluated with MTT assay. Protein removal efficacy of sponge brush product from the bovine serum albumin coated sheet was tested using bicinchoninic acid assay. The results of the length and diameter range of brush were 17~24 mm and 14~24 mm respectively.The length and thickness range of the handle was 14~16 cm and 3~5 mm respectively. There were significant differences in the sponge brush weight of all products (p<0.05), and porosity of the sponge was indicated to be more than 80% in all products.The water sorption rate ranged from 21 to 62% (p<0.05). As the results of extraction, there were neither foreign materials nor impurities in all of sponge brush products. The cell viability was indicated to be more than 70% except for two products.After applying all sponge brush products, the protein removal rate was more than 70%. The above results will provide useful information for future development of related oral care products.

The Past and Current Status of Dentists in Japan / 치위생과학회지

The purpose of this study is to provide the general history of fostering dentists in Japan and introducing their new roles. This research was conducted based on the government policy report on dentists and the information published by each educational institution. Based on the collected data, the official websites were used to represent the latest statistics of the institutions. The number of dentists in Japan has increased. The government established the National Examination for Dentists to guarantee the quality of dentists. After the standards for developing questions for the national examination were established in 1985, the contents of the examination have been appropriately improved by revising the standards every four years. This improvement has required dental students to study a variety of subjects for six years at dental school. Since dentists in Japan are required to respond to various demands from the nation; the Model Core Curriculum for Dental Education was developed to teach medical ethics and abilities to ensure that dentists conduct themselves professionally. Recently, the roles of dentists have been changing in Japan.When providing dental services to older patients over the age of 65, dentists and other dental professions focus on maintaining oral functions, such as saliva secretion, bite force, tongue movement, and masticatory/swallowing functions. However, oral function-related services for children are different. In addition to providing essential dental services, dental practitioners also provide special treatment, such as oral muscle training, myofunctional therapy, health guidance, and space retainers to the child patients with developmental insufficiency in oral functions. Dentistry in Japan has undergone numerous changes over the years and has continued to offer high-quality dental health services. Thus, information gained from the Japanese experience may be helpful to dental professions in other developed countries for planning oral health measures.

Relevance between Marketing Route of Social Media and Consumer Age Group for Choosing Dental Clinics / 치위생과학회지

Background@#The purpose of the study was to evaluate the relationship and route of dental Social Media marketing by age group and support effective dental marketingy by age group. @*Methods@#A study was conducted on 265 people, aged 20 to 64 years, who lived in Seoul, Gyeonggi area and regularly used one or more of the social media platforms, Naver Band, Facebook, Instagram, KakaoStory, Twitter, or YouTube more than once a day. A 27-question questionnaire survey of approximately 10 minutes was conducted, and the collected data was statistically analyzed using the PASW program, with the significane level set to 0.05. @*Results@#“Introduction of acquaintances” was the most common route to visit the dentist. Regarding the use of social media platforms based on age group, ‘Instagram’ had the highest frequency among people belonging to the age groups of 20 to 29 years and 30 to 39 years; ‘YouTube’ had the highest frequency among those aged 40 to 49 years; and ‘Naver Band’ had the highest frequency among those aged 50 to 65 years. @*Conclusion@#The most frequently used social media by consumers according to age included Facebook, YouTube, and Instagram. However, social media was found to have no significant impact on the choice of dental institutions, as the number of people who visited the dentist through “Introduction of acquaintances” was the highest, and “Introduction of acquaintances” did not have experience accessing the dentist site after dental marketing. If this study could provide customized marketing information for each age group through social media, it is expected that the marketing effect of dental institutions through social media would be maximized in the future.

Status of Oral Care according to the Type of Surgery for Inpatients at Dental Hospitals / 치위생과학회지

Background@#The aim of this study was to understand the importance of professional oral care for inpatients by examining the type and frequency of surgery and hospital discharge period at dental hospitals, and identify the types of professional oral care actually in progress. @*Methods@#In this study, the type of surgery and length of hospitalization were investigated among patients admitted to the dental hospital for oral and maxillofacial surgery, and the professional oral care status of inpatients who had difficulty self-managing their oral care was retrospectively identified by collecting data on oral care before and after surgery, including the type and frequency. @*Results@#The majority of inpatients at dental hospitals were male (57.6%), elderly patients over 60 years accounted for 20% of patients, and the average length of hospitalization was 4 days. In the 20s (aged 20∼29 y), the number of orthognathic surgery patients (73.1%) was high, and the incidence of cysts was high in middle-aged patients. Regarding the oral care of hospitalized patients, scaling was performed once by a dentist before surgery. After surgery, surgical dressings using H2O2 balls were applied and oral care education was introduced before discharge. @*Conclusion@#Based on the results of this study, professional oral care is essential to prevent infection and complications caused by oral bacteria among inpatients at dental hospitals. It is necessary to use various oral hygiene aids for inpatients and to conduct effective oral care instruction according to each patient’s situation. In addition, it is necessary to raise awareness and the role of dental hygienists in professional oral care.

The effect of general health status, oral health behavior and status on quality of life in Korea adults / 대한구강보건학회지

Objectives@#The purpose of this study was to analyze the relations between general health status, oral health behavior, oral health status, and quality of life, as well as the mediating effects of oral health behavior and oral health status. @*Methods@#Subjects aged ≥19 years participated in a health questionnaire in the third year of the Japanese National Health and Nutrition Examination Survey (2015). @*Results@#Analysis of the overall path of periodontal disease showed that respiratory and circulatory diseases and oral health behaviors had statistically significant indirect effects on quality of life through the mediation of oral health behavior and oral health conditions, while analysis of the overall trend of the decayed, missing, and filled teeth (DMFT) index showed significant indirect effects on quality of life through mediation between oral and oral health conditions. As a result of these analyses, the association of general health status with quality of life, and its effect thereon, were confirmed through the intermediary of oral health behaviors and oral health conditions. @*Conclusions@#Therefore, further research is needed on the relation of general diseases to oral health management. Special oral health departments need to be established in both general and university hospitals, and dental hygienists in charge of oral care need to be deployed to improve oral health for general diseases. Manuals and systems for oral health management and oral health promotion need to be developed for certified dental hygienists in Japan, specifically for each systemic disease. For example, hygienists should have guidance on the oral health practices that help in the prevention of diabetes.

Suggestions for the Work of Dental Hygienists and a Reflection of Health Insurance Charges according to Demographic Changes / 치위생과학회지

Given that the demand for dental care for elderly individuals is expected to increase according to demographic changes, long-term roadmaps for the current health insurance system should be actively planned. The study aimed to investigate the health insurance system in Korea, compared to that of Japan, and suggest directions for the work of dental hygienists to provide basic data on efficient improvements in the health insurance system. Based on the collected data, the website was used to collect additional related data from Korea and Japan. The most common cause of death in Korea was malignant neoplasms, and most patients were hospitalized for diseases and disorders of the musculoskeletal system and connective tissue. Dental care covered by Korean medical insurance for individuals aged 65 years or older was treatment oriented, such as dentures and implants, while in Japan, treatment-intensive items, such as visiting a dental hygienist for guidance and provision of home-care professional oral hygiene treatment, were covered. The percentages of remaining teeth in Korea and the percentage of remaining 20 teeth were 68.9% in those aged 65 to 74 years and 51.2% in those aged 75 to 84 years in Japan. A strategy for promoting oral health among elderly individuals should be established, and a multilateral intervention approach is required to prevent oral problems from leading to deterioration of whole-body health. The role of dental hygienists in providing comprehensive assessment to elderly individuals is important. In order to provide systemic oral care, it is necessary to introduce oral care systems according to the national policy.

Dental Hygienists in Japan / 치위생과학회지

The purpose of this study was to determine the status of dental hygienists in Japan. The study explicated the history, core curriculum, National Examination for Dental Hygienists, work roles of dental hygienists, and workplaces of dental hygienists. This study was based on the government policy report on dental hygienists and the information published by each public institution. The latest statistics presented by the institutions were collected through official websites. The employment information of graduates from Hiroshima University was analyzed based on actual field study. The results of the study revealed that social demand for dental hygienists has steadily increased and policies and education have been revised accordingly. The work roles of dental hygienists have expanded to meet the needs of the treatment and those of public health fields. In line with major policy changes, the educational period has been extended from 1 year to 3 or 4 years, while the mandatory credits for graduation have been established. Licensing examinations were being performed by the local governments since 1948 due to the different situations of dental hygienists in different areas. In 1992, they were converted into a single national examination. The work roles have expanded from assisting dental treatments to health guidance, home care, and perioperative care. Consequently, the number of dental hygienists has increased, especially in healthcare facilities for the elderly. Dental hygienists perform various roles. However, the most essential role is to provide the best oral care services to the patient. The expected role of dental hygienists has expanded in alignment with public healthcare needs and support for the elderly. The government and universities are expected to bring about improvements such as diversifying the channels of education and establishing policies to respond to growing patient needs by cultivating excellent dental hygiene professionals.

Elderly Welfare System and Role of Dental Hygienists in Korea and Japan / 치위생과학회지

In accordance with the aged society, oral health care for the elderly is considered important to maintain general health. Although the role of dental hygienists is essential for proper health management of the oral cavity, research on the care system for the elderly people's oral health and on the role of dental hygienists in the field of elderly welfare is still insufficient. Hence, the aim of this study is to investigate the status of Korean elderly welfare system and dental hygienists by comparing them with those in Japan, a precedent of aged society. First, we compared and investigated the Japanese long-term insurance system, which provides an institutional basis for a long-term care system for the elderly in Korea. Second, the elderly welfare law and care system, focusing on oral care, were examined. Lastly, in elderly care, we analyzed the distinctions between Korea and Japan regarding dental hygienists' role and scope of work. Taken together, as a precedent of aged society, Japan has shown well-specialized and systematic welfare for the elderly compared with Korea. With the development of the welfare system for the elderly in Japan, the role and the workscope of dental hygienists have been expanded to improve quality of life of elderly people, as a key professional for elderly oral care. Therefore, we should perceive the need for improvement of long-term care insurance and the expansion of dental hygienists' work in Korea. In conclusion, these results could be used as basic data for improving the elderly welfare system and developing dental hygienists in Korea.

Hydrogen peroxide inhibits Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase in mouse parotid acinar cells

Intracellular Ca²⁺ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H₂O₂) on cytosolic Ca²⁺ accumulation in mouse parotid acinar cells. Intracellular Ca²⁺ levels were slowly elevated when 1 mM H₂O₂ was perfused in the presence of normal extracellular Ca²⁺. In a Ca²⁺-free medium, 1 mM H₂O₂ still enhanced the intracellular Ca²⁺ level. Ca²⁺ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H₂O₂. On the other hand, 10 mM H₂O₂ induced more rapid Ca²⁺ accumulation and facilitated Ca²⁺ entry from extracellular fluid. Ca²⁺ refill into intracellular Ca²⁺ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca²⁺ release from Ca²⁺ store was not affected by 1 mM H₂O₂ in permeabilized cells. Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase (PMCA) was markedly blocked by 1 mM H₂O₂ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H₂O₂-induced Ca²⁺ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H₂O₂ under pathological conditions may lead to cytosolic Ca²⁺ accumulation and that the primary mechanism of H₂O₂-induced Ca²⁺ accumulation is likely to inhibit Ca²⁺ efflux through PMCA rather than mobilize Ca²⁺ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.

Activation of calcium signaling by hepatitis B virus-X protein in liver cells

Hepatitis B virus x gene product (HBx) is known to be a transactivator of transcriptional elements that regulate the expression of a variety of genes associated with the growth, differentiation, survival and the apoptosis of cells. However, the exact mechanism of the activation and inhibition of cellular events by HBx remains uncertain. The present study was designed to measure the effect of HBx, on the signal transduction pathways associated with intracellular Ca(2+)mobilization following HBx transfection in the stable Chang liver cells (CHL-X). Enhanced cell proliferation by HBx in CHL-X was confirmed by MTT assay and by the immunodetection of PCNA. The transactivation of AP-1 by HBx induced in CHL-X was inhibited by cyclosporin A (CsA), a mitochondrial Ca(2+)channel blocker and by BAPTA-AM, a cytosolic Ca(2+)blocker. Activation of the SAPK/JNK signaling pathway by HBx was evidenced by the increased phosphorylations of c-Jun (Ser63) and of JNK (Thr183/Tyr185). Increased phospho-Erk/Erk and phospho-Raf1/Raf in HBx-induced CHL-X indicated that HBx might stimulate the MAPK pathway. PI3K activity and cytosolic free Ca(2+)levels were elevated in HBx-induced CHL-X. These results imply that HBx transactivates both JNK and MAPK signal transduction pathways in association with the mobilization of cytosolic Ca(2+).

Expression and characterization of a mutant recombinant blood coagulation factor VIII (rFVIII (m))

In an earlier study, a site directed mutant rFVIII (rFVIII(m), Arg(336) -> Gln(336)) expressed in baculovirus-insect cell (Sf9) system was found to sustain high level activity during incubation at 37 for 24 h while the cofactor activity of normal plasma was declined steadily. In this study, a mutant B-domain deleted rFVIII(m), Arg(336) -> Gln(336) expressed in baculovirus-insect cell (Sf9) system was characterized for its enzymatic and chemical properties. The expressed rFVIII(m) and plasma FVIII (pFVIII) were purified by immunoaffinity column chromatography and identified by Western blot analysis. The partially purified rFVIII(m) exhibited cofactor specific activity of 2.01 X 10(3)units/mg protein. The molecular weight of rFVIII(m) ranged between 40 to 150 kDa with a major band at 150 kDa. Treatment of both rFVIII(m) and pFVIII with thrombin increased their cofactor activity in a similar pattern. Treatment of both the activated rFVIII(m) and native FVIII with APC decreased their cofactor activities, however, the former exhibited a slower decrease than the latter, although no significant difference was present. rFVIII(m) formed a complex with vWF, resulting in a stabilized form, and the lag period of thrombin-mediated activating was extended by vWF association. These results implicated that rFVIII(m) expressed in baculovirus-insect cell system had a comparable capacity as FVIII cofactor activity and might be a good candidate for the FVIII replacement therapy for hemophilia A patients.

Regulation of leptin gene expression by insulin and growth hormone in mouse adipocytes

The role of leptin in the control of obesity, insulin resistance and type II diabetes has been reported, however, the regulatory mechanism of leptin in animals affected by hormones is not clearly understood. In this study, the effects of insulin, epinephrine, growth hormone or dexamethasone on the expression of leptin was examined in mouse primary adipocytes. The leptin expression was also studied in the adipose tissue of the mouse treated with insulin or growth hormone (0.3 or 0.6 units/animal). Insulin (100 nM) or dexamethasone (100 nM) stimulated leptin mRNA transcription while epinephrine (100 nM) alleviated its transcription in mouse primary adipocytes. The level of leptin protein in cultured media of adipocytes treated with insulin or dexamethasone was higher than that of the control group but growth hormone or epinephrine treatment had no effect on them. Insulin administration (0.6 units/mouse) enhanced leptin mRNA as well as leptin protein in mouse adipose tissue but growth hormone administration (0.3 or 0.6 units/mouse) had no effect on them. Leptin protein level in sera of mice injected with insulin or growth hormone was not significantly different from that of control group. These results indicate that both insulin and dexamethasone stimulate leptin gene expression and secretion of its product, whereas, growth hormone has no effect on the expression of leptin gene in mouse adipocytes.

Regulation of leptin gene expression by insulin and growth hormone in mouse adipocytes

The role of leptin in the control of obesity, insulin resistance and type II diabetes has been reported, however, the regulatory mechanism of leptin in animals affected by hormones is not clearly understood. In this study, the effects of insulin, epinephrine, growth hormone or dexamethasone on the expression of leptin was examined in mouse primary adipocytes. The leptin expression was also studied in the adipose tissue of the mouse treated with insulin or growth hormone (0.3 or 0.6 units/animal). Insulin (100 nM) or dexamethasone (100 nM) stimulated leptin mRNA transcription while epinephrine (100 nM) alleviated its transcription in mouse primary adipocytes. The level of leptin protein in cultured media of adipocytes treated with insulin or dexamethasone was higher than that of the control group but growth hormone or epinephrine treatment had no effect on them. Insulin administration (0.6 units/mouse) enhanced leptin mRNA as well as leptin protein in mouse adipose tissue but growth hormone administration (0.3 or 0.6 units/mouse) had no effect on them. Leptin protein level in sera of mice injected with insulin or growth hormone was not significantly different from that of control group. These results indicate that both insulin and dexamethasone stimulate leptin gene expression and secretion of its product, whereas, growth hormone has no effect on the expression of leptin gene in mouse adipocytes.

Retroviral - mediated Transduction of Leptin Gene in Genetically Obese Mice / 대한내분비학회지

BACKGROUND: Leptin gene is known to be related to obesity in human and animals and complete genetic defect of the gene in ob/ob mouse has been identified. Therefore, ob/ob mouse is widely used as an animal model for the study of etiology and therapy of obesity. The main biological function of leptin was thought to involve in the regulation of food intake and weight gain, however, the regulatory mechanisms by which leptin functions in the weight reduction and lowering the blood glucose level are uncertain. In the present study, retroviral-mediated leptin gene transduction into ob/ob mouse was attempted for the correction of biochemical parameters of obesity. METHODS: Leptin cDNA was inserted into pLXSN retroviral vector (pLXSN-lep) and recombinant leptin expressing retrovirus particles (3 X10 CFU/mL) were produced in psi2 ecotropic packaging cells and subsequent transfection into PA317 amphotropic packaging cells. The leptin expressing recombinant viruses (LER) were transduced into NIH3T3 mouse fibroblasts and insertion of leptin cDNA into chromosomal DNA of PA317 and MH3T3 mouse fibroblasts was confirmed by Southern blot hybridizations. Leptin mRNA and its protein expressed in the cells were identified by Northern blot hybridization and Western blot immunodetection method, respectively. LER were injected I. P. into ob/ob mice, and body weight, food intake, serum leptin level and blood glucose level were measured. RESULTS: Expression of leptin was identified in PA317 and NIH3T3 mouse fibroblasts transduced with LER. Leptin content in sera of mice transfused with LER was drastically increased after 1 week and decreased to the almost basal level at 3 weeks after the transfusion. The body weight as well as food intake of ob/ob mouse transduced by LER decreased for the first 3 weeks and slightly increased thereafter. The reduction of both body weight and food intake in ob/ob mice transduced with LER was observed with the concomitant increase of serum leptin level, indicating that retroviral-mediated transduction of leptin gene in ob/ob mouse in vivo produced a biologically active leptin protein and released it into blood circulation. CONCLUSION: A transient expression of leptin cDNA in ob/ob mice by a retroviral-mediated transduction was performed and further studies are required for long term expression of the gene in vivo.

Purification of the major allergens from Korean Dermatophagoides pteronyssinus and production of the recombinant antigens / 천식및알레르기

BACKGROUND: Purified major allergens of house dust mite are essential for evaluation of the allergic mechanism in molecular basis and development of new modalities of immunemodulation. OBJECTIVE: In this study, we aimed to purif group 1 and group 2 allergens from Dermatophagoides pteronyssinus (Dp). In addition, cDNAs corresponding to Der pI and II in Korean Dp were isolated and recombinant Der p1 and Der pII were synthesized. MATERIALS AND and METHOD: Der pI allergen was purified by ammonium sulfate precipitation, anion -exchange column chromatography, and gel filtrat,ion chromatography. Der pII allergen was purified by anion exchange chromatography, gel filtration chromatography, and a preparative isoelectric focusing method. RESULTS: Eight hundred ug of Der pI and 50 ug of Der pII were obtained from 100 g of culture medium and 1 g of mite bodies, respectively. The purities of these allergens were confirmed by SDS PAGE and the strong reactivity to the patient sera was identified. In order to produce a recombinant allergens, poly(A) RNA from house dust mites were isolated and used for cDNA synthesis by RT PCR. The cDNA was inserted into prokaryotic expression vector and the vectors were transformed into E. coli. A little amount of recombinant Der pI protein was produced due to the low solubility, and 1.2 mg of recombinant Der pII was produced from 1 L of E. coli culture medium. The antigenicity of Der pI was relatively weak, however, Der pII showed a strong antigenicity. Amino acid sequence of the amplified cDNA deduced from DNA sequences of Der pII showed 6 different variants. The variation of amino acid sequences suggests the possibility of high incidence of mutation of Der pII protein. CONCLUSION: A simplified method for the purification of Der pI and Der pII was developed. Recombinant allergens will be useful for the diagnosis and treatment of allergy with lower costs.

Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII

FVIII is synthesized as a single chain precursor of approximately 280 kD with the domain structure of A1-A2-B-A3-C1-C2 and it circulates as a series of metal ion-linked heterodimers that result from cleavages at B-A3 junction as well as additional cleavages within B domain. Factor VIII is converted to its active form, factor VIIIa, upon proteolytic cleavages by thrombin and is a heterotrimer composed of the A1, A2, and A3-C1-C2 subunits. A1 subunits of factor VIIIa terminates with 36 residue segment (Met337-Arg372) rich in acidic residues. This segment is removed after cleavages at Arg336 by activated protein C, which results in inactivation of the cofactor. In the present study, site-directed mutagenesis of FVIII at Arg336 to Gln336 was performed in order to produce an inactivation resistant mutant rFVIII (rFVIIIm) with an extended physiological stability. A recombinant mutant heavy chain of FVIII (rFVIII-Hm; Arg336 to Gln336) and wild-type light chain of FVIII (rFVIII-L) were expressed in Baculovirus-insect cell (Sf9) system, and a biologically active recombinant mutant FVIII (rFVIIIm) was reconstituted from rFVIII-Hm and rFVIII-L in the FVIII-depleted human plasma containing 40 mM CaCl2. The rFVIIIm exhibited cofactor activity of FVIIIa (2.85 x 10(-2) units/mg protein) that sustained the high level activity during in vitro incubation at 37 degrees C for 24 h, while the cofactor activity of normal plasma was declined steadily for the period. These results indicate that rFVIIIm (Arg336 to Gln336) expressed in Baculovirus-insect cell system is inactivation resistant in the plasma coagulation milieu and may be useful for the treatment of hemophilia A.

Prokaryotic expression and characterization of human AP DNA endonuclease

The expression of major human apurinic/apyrimidinic DNA endonuclease (APEX) from its cDNA in E. coli (DH5 alpha) was attempted in order to obtain a biologically active recombinant APEX. E. coli cells were transformed by a prokaryotic translation vector (pGEX-4T-3) harboring APEX cDNA. GST-APEX fusion protein with a molecular weight of 6.3 KDa was induced by IPTG (1.0 mM) treatment. Western blot immunodetection identified the induced protein as the GST-APEX fusion protein. The survival rate of E. coli cells (DH5 alpha) transformed with pGEX-4T-3-APEX increased when the cells were treated with N-diethyl-N-nitrosamine (DENA) or 3'-methyl-4-monomethylaminoazobenzene (3'-MeMAB), indicating that APEX expression had a protective effect on the cytotoxicity of these carcinogens. The fusion protein extracted from E. coli cells and purified by GSH-agarose gel affinity chromatography exhibited APEX activity. Treatment of thrombin to the GST-APEX fusion protein and affinity purification followed by Sephacryl S-100 gel filtration resulted in APEX peptide with MW 36 KDa, which exhibited AP DNA repair activity (8,7000 EU/mg protein). N-ethylmaleimide (0.1 mM) or AMP (0.98 mM) inhibited APEX activity by 50% and kinetic analysis indicated that the recombinant APEX (rAPEX) had a Km value of 0.022 microM (AP sites for AP DNA) and the Ki value was 0.48 mM for AMP. These results indicated that E. coli cells expressing biologically active GST-APEX were resistant to the cell damage caused by chemical carcinogens and that rAPEX purified from E. coli cells transformed with APEX cDNA-inserted translation vector was similar to native APEX in some properties.

Adaptive Change of AP DNA Endonuclease Against Genotoxic Agents in Normal and Transformed Cells / 대한암학회지

PURPOSE: AP DNA endonuclease (APE), an enzyme responsible for the repair of damaged DNAs, is essential for the maintenance of genetic information of cells. Deficiency of APE in certain hereditary skin tumor and senescent cells has been implicated but the regulation of APE activity as well as the expression of APE gene in response to DNA damage has not been well documented. Genotoxic agents including ultimate carcinogens that can damage DNA were treated to cultured normal and transformed human cells and adaptive response of APE gene expression to these treatments was measured in order to evaluate the role of APE in chemical carcinogenesis. MATERIALS AND METHODS: Hydroxyl radical ('OH) generated from H2O2 (60 uM) through Fenton reaction, each 100 uM of N-nitrosomethylurea (NMU), 3-methyl-4-monomethyl- aminoazobenzene (3'-MeMAB) and N-acetoxy-2-acetaminofluorene (AAAF) were treated to umbilical cord blood cells (UCBC), HepG2 cells and HL-60 cells. APEX mRNA and APEX protein contents expressed in these cells exposed to each of these agents were measured by Northern blot hybridization and Western blot immunodetection analysis. The changes of APE activity in cells exposed to these genetoxic agents were measured. RESULTS: Treatment of H2O2 (60 uM) to UCBC, HepG2, and HL-60 cells increased APE activity significantly and pretreatment of a catalytic agent for OH, FeSO4 (60 pM) to the cells prior to H2O2 exposure did not further increase the APE activity in cells. Adaptive response to H2O2 in HL-60 cells increased in proportion to the concentration of H2O2 up to 60 pM. However, further increase in H2O2 concentration had no effect on the enzyme activity. Treatment of NMU (100 pM), 3-MeMAB (100 pM) and AAAF (100 pM) to these cells brought about a slight increase in the APE activity. APEX mRNA expression in UCBC and HepG2 cells exposed to H2O2, NMU, 3-MeMAB was markedly increased in APEX mRNA expression. APEX mRNA expression was also increased in HL-60 cells exposed to H2O2 (60 pM) and 3-MeMAB (100 uM) but NMU (100 pM) exposure to the cells resulted in a slight increase of it (Fig. 2). APEX protein expression was increased in all UCBC, HepG2 and HL-60 cells exposed to these genotoxic agents (Fig. 3). CONCLUSION: These results implicate that exposure of genotoxic agents to the cultured cells may cause DNA damage and lead to adaptive increase in APE activity as well as APE gene expression. It is probable that APE gene is transcriptionally regulated in response to the exposure of H2O2 or 3-MeMAB in cultured human cells as a consequence of activation of DNA repair system for the adaptation to the crisis.