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Objective This study aimed to compare the prognostic utility of sentinel node biopsy and elective neck dissection in early stage clinically node-negative oral cavity squamous cell carcinoma patients. Method PubMed, Scopus, Embase, Web of Science and Cochrane Library databases were searched up to March 2022. Hazard ratios, Kaplan–Meier curves, p-values and survival outcomes were extracted. Results Twelve studies involving 10 583 patients were included. No significant differences in overall survival between sentinel node biopsy and elective neck dissection groups were found. Heterogeneity was not detected in pooled overall survival, disease-free survival and disease-specific survival analyses (all I2 less than 50). In subgroup analyses by follow-up period, sentinel node biopsy and elective neck dissection had similar prognostic value. Conclusion Sentinel node biopsy might be a valuable alternative to elective neck dissection for the management of early stage clinically node-negative oral cavity squamous cell carcinoma.

This study aimed to investigate the relationship between hormone replacement therapy (HRT) types and breast cancer (BC) incidence in postmenopausal women in Korea. The nested case–control study used data from the National Health Insurance Service database. Among the women aged ≥50 years who menopaused between 2004 and 2007, BC incidence up to 2017 was analyzed in 36,446 women using or having used HRT for >1 year and in 36,446 women who did not use any HRT for more than 1 year. HRT types and duration were classified into three categories. BC risk (BCR) decreased with tibolone use for all ages. With HRT initiation in women aged ≥50 years, BCR was lower with tibolone and estrogen–progestogen therapy. HRT for 5 years than in the control group. For women in their 50s, tibolone use lowers BCR; for all ages, the use of any HRT for >5 years showed lower BCR in Korea. These divergent results from western countries could be associated with the specific characteristics of BC in Korea.

BACKGROUND: Long-term mortality following tuberculosis (TB) diagnosis in Korea remains unclear.METHODS: The present study used data from the National Health Insurance Service database, an extensive health-related database including most Korean residents. TB patients were identified using International Classification of Diseases, Tenth Revision coding (A15-19, U88.0-88.1) and the type of anti-TB drug(s) between 2003 and 2016. Long-term mortality and causes of death in TB patients were analysed.RESULTS: A total of 357 211 individuals had TB over the period from 2003 to 2016 and 103 682 died. The mean age of the cohort was 54.7 ± 20.7 years, and 59.8% were male. The survival probability of TB patients at 1, 5, and 10 years after diagnosis was 87.8%, 75.3%, and 63.3%, respectively. High mortality and TB-related death rates were especially prominent in the early stages after TB diagnosis. The overall standardized mortality ratio of TB patients to the general Korean population was 3.23 (95% confidence interval 3.21–3.25).CONCLUSION: Mortality in TB patients was especially high in the early stages of disease after TB diagnosis, and mostly due to TB. This figure was approximately three-times higher than the mortality rate in the general population.

Supplemental material, Supplementary Figure and tables for Urinary vitamin D-binding protein, a novel biomarker for lupus nephritis, predicts the development of proteinuric flare by D J Go, J Y Lee, M J Kang, E Y Lee, E B Lee, E C Yi and Y W Song in Lupus

A compact surface potential based model for organic thin-film transistors (OTFTs), including both tail and deep trap states across the band gap, is reported. The model has been developed on the basis of a complete surface potential approach for undoped-body OTFTs. Accurate surface potentials are calculated by explicitly including the floating backside potential that varies with applied biases. A pseudo-2D resistor model is developed to capture the structural features of the OTFT. The resistor model considers, in particular, the effects originating from a bias dependent 2D current flow in the channel region and results in accurate reproduction of the electrical characteristics. The fitting capability of the developed OTFT model is verified against measured high-performance dinaphtho thieno thiophene (DNTT) based field-effect transistor data. Accurate reproduction of the current characteristics of the OTFT test structures is verified from a week to a strong inversion regime.

Genomic reprogramming factors in the cytoplasm of germinal vesicle (GV) stage oocytes have been shown to improve the efficiency of producing cloned mouse offspring through the exposure of nuclei to a GV cytoplasmic extract prior to somatic cell nuclear transfer (SCNT) to enucleated oocytes. Here, we developed an extract of GV stage pig oocytes (GVcyto-extract) to investigate epigenetic reprogramming events in treated somatic cell nuclei. This extract induced differentiation-associated changes in fibroblasts, resulting in cells that exhibit pluripotent stem cell-like characteristics and that redifferentiate into three primary germ cell layers both in vivo and in vitro. The GVcyto-extract treatment induced large numbers of high-quality SCNT-generated blastocysts, with methylation and acetylation of H3-K9 and expression of Oct4 and Nanog at levels similar to in vitro fertilized embryos. Thus, GVcyto-extract could elicit differentiation plasticity in treated fibroblasts, and SCNT-mediated reprogramming reset the epigenetic state in treated cells more efficiently than in untreated cells. In summary, we provide evidence for the generation of stem-like cells from differentiated somatic cells by treatment with porcine GVcyto-extract.

In this study, we examined whether Hanganutziu-Deicher (H-D) antigens are important as an immunogenic non-α1,3-galactose (Gal) epitope in pigs with a disrupted α1,3-galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells. A total of 1800 somatic cell nuclear transfer (SCNT) embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. The α1,3-galactosyltransferase activity in lung, liver, spleen, and testis of heterozygote α1,3-galactosyltransferase gene knockout (GalT-KO) pigs was significantly decreased, whereas brain and heart showed very low decreasing levels of α1,3-galactosyltransferase activity when compared to those of control. Enzyme-linked lectinosorbent assay showed that the heterozygote GalT-KO pig had more sialylα2,6- and sialylα2,3-linked glycan than the control. Furthermore, the heart, liver, and kidney of the heterozygote GalT-KO pig had a higher N-glycolylneuraminic acid (Neu5Gc) content than the control, whereas the lung of the heterozygote GalT-KO pig had Neu5Gc content similar to the control. Collectively, the data strongly indicated that Neu5Gc is a more critical xenoantigen to overcoming the next acute immune rejection in pig to human xenotransplantation.

Preoperative planning, or intraoperative navigation of hip surgery, including joint-preserving procedures such as osteotomy or joint-replacing procedures such as total arthroplasty, needs to be performed with a high degree of accuracy to ensure a successful outcome. The ability to precisely localise the hip joint rotation centre may prove to be very useful in this context. The human hip joint has been shown to be a conchoid shape, and therefore the accurate location of the hip joint centre (HJC) cannot be computed simply as the centre of a sphere. This study describes a method for determining the HJC by applying a conchoid shape to the acetabular cartilage surface of magnetic resonance images, in order to increase the accuracy of the HJC location which had previously been calculated by a functional method using reconstructed three-dimensional surface bony models. By approximating a conchoid shape to the acetabulum, it was possible to compensate for HJC calculation errors.

We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) andα2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level ofα-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production ofN-glycolylneuraminic acid (Neu5Gc) due to an increase ofα2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.

Background and purpose: Intrahospital delay is the most serious obstacle in thrombolysis in acute ischaemic stroke (AIS). We implemented the pre-hospital notification system from the emergency medical information system in our metropolitan area to reduce intrahospital delay. Methods: From October 2007, we implemented a 24-h hotline system between our stroke center and the Korean Emergency Medical Information System in Busan. We compared processing times and clinical outcomes amongst patients after using intravenous tissue type plasminogen activator (iv t-PA) with and without the hotline system. Results: After the pre-hospital notification system was implemented, the rate of iv t-PA use increased from 6.5% to 14.3%. Time of onset in patients with pre-hospital notification was much longer than in patients without (121.5 ± 34.8 min vs. 74.7 ± 38.5 min, P

CCAAT/enhancer binding protein a (C/EBPα) plays an important role in lipid deposition and adipocyte differentiation. In order to find genetic markers to improve the meat quality of Korean cattle, the bovine C/EBPa gene was chosen as a candidate gene to investigate its association with carcass and meat quality traits in Korean cattle. A single nucleotide polymorphism (SNP) was identified at position 271 (A/C substitution) of coding region in the C/EBPa gene. A PCR-RFLP procedure with restriction enzyme Smal was developed for determining the marker genotypes. The frequencies of alleles C and A and were 0.374 and 0.626, respectively. The genotype frequencies for CC, AC and AA were 12.9, 49.0 and 38.1%, respectively, in Korean cattle population. The frequencies of genotype were in agreement with Hardy-Weinberg equilibrium. Association analysis indicated that the gene-specific SNP marker of C/EBPa showed a significant association with marbling score (p

A sensitive and specific assay was developed to detect potato blackleg caused by Pectobacterium atrosepticum in potato. Primers PEAF and PEAR from the Rhs family gene homologous to RhsA (cell envelope biogenesis – outer membrane) were used to amplify a 904 bp DNA fragment. PCR was used to detect the pathogen in artificially inoculated potato seed tubers. The PCR product was only produced from 12 isolates of P. atrosepticum from various countries among 36 isolates of other species of Pectobacterium, Pseudomonas, Xanthomonas, as well as Escherichia coli and the soilborne fungus Fusarium oxysporum f.sp. dianthi.

A synthetic layered double hydroxide (LDH: Mg-Al-CO3) was heated to 560 °C for 3 h in air, and the resulting calcined LDH (1.00 g dm−3) was used in the sorption study of aquatic CrO4 2− (concentration range: 1.0 × 10−3 − 5.0 × 10−3 mol dm−3) under CO2 free atmosphere. Reconstructed LDH containing intercalated CrO4 2− between interlayers was formed and this was confirmed by X-ray powder diffraction (XRD), 27Al MAS NMR, FT-IR and UV/VIS spectroscopies. The sorption of CrO4 2− by LDH resulted in increased hydroxide concentration, suggesting that ion-exchange based mechanism is involved: LDH + CrO4 2− = LDH(CrO4)+ 2OH−. The equilibrium constant (K), for sorption of CrO4 2−, based on ion-exchange with two OH− per unit LDH (composition: Mg6Al2(OH)18), of 3.1 ± 0.4 mol dm−3 was evaluated by a non-linear least squares fit procedure.

In this paper, a surface potential based compact model for organic thin-film transistors (OTFTs) including both tail and deep trap states across the band gap is presented and benchmarked against measured data from high-performance dinaphtho thieno thiophene (C10-DNTT) based test devices. This model can accurately describe the OTFT test-structure current from week to strong inversion regime.

Optimum shape design of the tool structure is carried out in order to decrease the deformation and the stress from the large amount of stamping load with a simultaneous effect of weight reduction. Topology optimization is carried out to design the shape of the rib structure and Taguchi method is utilized to optimize the core shape. As a result of optimization, the weight of the rib and the core structures is reduced to 3.1% and the deformation and the stress of the rib and the core structures are decreased to 10.6% and 3.7% comparing to the initial design, respectively.

The thermal effect and athermal effect such as electro-plastic effect of metallic materials induced by high density current can dramatically reduce the flow stress, which is beneficial to the forming process of less formable metal. In this paper, pulse current-assisted deep drawing of the magnesium alloy is proposed due to lower energy consumption and higher efficiency. In this process, the metal sheet is designed in series in a pulse current circuit and heated directly by the pulse current. In addition, the insulated mould is employed to avoid the current leaking. Experiments were conducted to demonstrate the feasibility and advantages of the proposed process. An experimental process system was established and the electrical-assisted Erichsen cupping tests and rectangular cup drawing tests were performed. The experiments showed that the forming load was reduced and the cupping height and associated principal strains were increased in the Erichsen cupping and deep drawing process assisted by high-density electric current.

SUMMARY The effects of water deprivation for 48 h on the pharmacokinetics and pharmacodynamics of furosemide were examined after intravenous, 8 mg/kg body weight, and oral administration, 16 mg/kg body weight, of furosemide to control and water deprived rats. After i.v. administration, the total body and nonrenal clearances decreased significantly in water–deprived rats. The urine output, urinary excretion of sodium, potassium and chloride based on grams of kidney weight, and the diuretic, natriuretic and chloruretic efficiencies decreased significantly in waterdeprived rats after both intravenous and oral administration of furosemide, suggesting that the dose of furosemide for water–deprived patients may require modification.

It is very important to investigate hemodynamic and hemorheologic features of blood flow from fluid mechanical point of view because they play major roles in understanding the pathogenesis on cardiovascular disorders. Generally, hemorheologic characteristics of blood flow near the vessel wall are well-known as the most important factor in thrombosis generation according to several hypotheses. To investigate the hemodynamic and hemorheological features related to circulatory diseases, in-vitro experiments were carried out using Micro-PIV technique. Numerical simulation methods using conventional CFD codes were also used to compare with experimental results. The vessel models with stenotic lesion and clinical stent implantation were made of PDMS channel based on Right Coronary Artery (RCA) from clinical angiogram for the patient with stenotic lesion. The hemodynamic and hemorheologic behaviors for control volume near stenotic lesion were evaluated by velocity profiles.

Intrahospital delay is the most serious obstacle in thrombolysis in acute ischaemic stroke (AIS). We implemented the pre-hospital notification system from the emergency medical information system in our metropolitan area to reduce intrahospital delay.From October 2007, we implemented a 24-h hotline system between our stroke center and the Korean Emergency Medical Information System in Busan. We compared processing times and clinical outcomes amongst patients after using intravenous tissue type plasminogen activator (iv t-PA) with and without the hotline system.After the pre-hospital notification system was implemented, the rate of iv t-PA use increased from 6.5% to 14.3%. Time of onset in patients with pre-hospital notification was much longer than in patients without (121.5 +/- 34.8 min vs. 74.7 +/- 38.5 min, P0.01) notification but door-to-needle time was significantly reduced (28.9 +/- 11.4 min vs. 47.7 +/- 22.8 min, P0.01). However, there were no significant differences in 90-day clinical outcomes between the two groups.The pre-hospital notification system reduced intrahospital processing times which led to increased iv t-PA use after AIS. However, the improvement of clinical outcomes in thrombolysis might require organization of not only intrahospital processes but of outside processes such as the early recognition and rapid dispatch of patients with suspected AIS.

Background and purpose: Aspirin is an important therapeutic regimen to prevent the recurrent ischemic events or death after acute ischemic stroke. In this study, we evaluated the relationship between the extent of adenosine diphosphate (ADP) -induced platelet aggregation and outcome in acute ischemic stroke patients on aspirin therapy. Methods: We selected 107 acute ischemic stroke patients who had been prescribed aspirin and evaluated platelet function test by using optic platelet aggregometer test after 5 days of taking it and investigated the prognosis 90 days after ischemic events. Kaplan–Meyer curve was used for survival analysis. Results: After stratification of the subjected patients by tertiles of ADP-induced platelet aggregation, the events rates were 7.4%, 9.3% and 30.8% (P = 0.023). In multiple logistic regression analysis, old age over 70 years (OR, 13.7; 95% CI, 2.14–88.07; P = 0.001) and the increased ADP-induced platelet aggregation had independent significance to the risk of primary end-points after acute ischemic stroke (OR, 1.1; 95% CI 1.01 to 1.20; P = 0.026). Conclusions: This study showed that the increased ADP-induced platelet aggregation under using aspirin is associated with poor outcome after acute ischemic stroke.

In this paper, we will present the results of WDM-PON based FTTH field trial test which held in the city of Gwangju. We have implemented injection locked FP-LD based WDM-PON system and reliably delivered IP-TV, N-PVR, HD-VoD, EoD and Internet service as FTTH service through the system during the field trial test. We have also verified that the WDM-PON system worked well to provide QoS guaranteed 100Mbps bandwidth per subscriber. Furthermore, we have presented network designing issues in OSP and CPN that should be overcome to efficiently deploy FTTH service. Finally, based on the field trial test results, we proposed FTTH service deployment strategies.

Case report. To describe a rare case of oncocytoma arising from the spinal cord in a 40-year old woman. Republic of Korea. The patient's history, physical examination, radiological and pathological findings were reviewed. A 40-year-old woman presented with 3-month history of low back pain. Magnetic resonance imaging revealed an intradural extramedullary mass located between L1 and L4. She refused any surgical treatment and so was discharged. At 10 days after discharge, an emergency operation was performed because of sudden paralysis in both lower extremities. The confirmed diagnosis is oncocytoma. At 4 months after surgery, the patient failed to obtain neurological recovery from complete paraplegia. Since the progression of an intradural extramedullary mass that shows minor neurological symptoms can lead to complete paraplegia in a short time, close observation and early surgical decompression are necessary.

To assess the effects of soybean-derived pinitol on glycaemic control and cardiovascular risk factors in Korean patients with type II diabetes mellitus.Randomized, double-blind, placebo-controlled, parallel-group trial.Pusan Paik Hospital, Pusan, Republic of Korea.A total of 30 patients with type II diabetes received an oral dose of 600 mg soybean-derived pinitol or placebo twice daily for 13 weeks.Pinitol significantly decreased mean fasting plasma glucose, insulin, fructosamine, HbA1c, and the homeostatic model assessment insulin resistance index (HOMA-IR, P0.001). Pinitol significantly decreased total cholesterol, LDL-cholesterol, the LDL/HDL-cholesterol ratio, and systolic and diastolic blood pressure and increased HDL-cholesterol (P0.05).These data suggest that soybean-derived pinitol may be beneficial in reducing cardiovascular risk in Korean type II diabetes.

To estimate the utility of thyroid transcription factor-1 (TTF-1) and the combined cytokeratin 7 (CK7) and 20 (CK20) immunoprofile as a marker for identifying the primary site of metastatic adenocarcinoma in effusions of the serous cavity.Formalin-fixed, paraffin-embedded cell block specimens of pleural and peritonealfluid diagnosed as metastatic adenocarcinomas with known sites of origin were used for TTF-1, CK7 and CK20 immunohistochemistry. The primary sites of these cases were lung (16 cases), ovary (15), stomach (9), colon (8) and breast (8) and were confirmed by radiologic and/or histologic evaluation.The lung adenocarcinomas showed TTF-1 positivity in 81% (13/16) of cases. All nonpulmonary adenocarcinomas lacked TTF-1 staining. The CK7-/CK20+ immunophenotype was seen in 63% of colonic adenocarcinomas and not seen in lung, ovary, stomach or breast adenocarcinomas. The CK7+/CK20- immunophenotype was seen in 100%, 88% and 87% of cases that originated in the lung, breast and ovary, respectively.TTF-7 immunostaining is useful in the differentiation between pulmonary and nonpulmonary origin of adenocarcinomas in malignant effusions. The combination of CK7-/CK20+ immunostaining is useful in identifying colon adenocarcinomas.

Nach einem auserhalb der Klinik stattgefundenen Herz-Kreislauf-Stillstand und zunachst erfolgreicher Reanimation sind die endgultigen Uberlebensraten und – bei den Uberlebenden – die neurologischen Ergebnisse weiterhin unbefriedigend. Dabei bleibt es schwierig, fruh das Outcome zu prognostizieren, da zuverlassige Marker fehlen. Ob die Serumlaktatkonzentration dabei hilfreich sein konnte, hat eine japanische Arbeitsgruppe untersucht.

Sample preparation methods and data acquisition protocols were optimized for the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to high-throughput quantitative analysis of low molecular mass substrates and products of an enzyme-catalyzed reaction. Using a deuterlum-labeled internal standard, precise standard curves were obtained (r(2) = 0.9998) over two orders of magnitude of concentration of rac-1-phenylethylamine (PEA), which is converted to 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) by a lipase-catalyzed reaction with ethylmethoxyacetate (EMA) as second substrate. Reliable relative standard deviations were achieved (or =5%) using automated analysis with peak intensity ratios between 0.2 and 5 of analyte to internal standard. This method permitted quantitative analysis of the lipase reaction, producing results comparable to those from gas chromatographic (GC) analysis in the dynamic range of GC. This work shows that MALDI-TOFMS can be applied for the high-throughput screening of enzymes.

Methylchlorogenic acid (MC) is one of the main components in the leaves of Eriobotrya japonica. We previously reported that MC is the most potent antioxidant among several components of Eriobotrya japonica, and its antioxidant activity is stronger than that of chlorogenic acid. Antioxidants are expected to inhibit redox-sensitive NFkappaB activation since NFkappaB is readily influenced by cellular oxidative state. Based on these findings, in vivo experiments with MC were conducted to determine its ability to downregulate the NFkappaB activation in mouse liver. Results clearly showed that MC is a potent suppressor of BHP-induced NFkappaB activation. We observed a significant reduction by MC on BHP-induced translocation of p65 subunit of NFkappaB. This may be due to formation of p50/p65 heterodimer, which is mainly inducible NFkappaB. MC slightly blocked the BHP-induced IkappaB alpha degradation. There is a possibility of IkappaB alpha resynthesis via activated NFkappaB during a 5 h waiting period following BHP injection. The present results suggest that MC may inhibit NFkappaB activation, exhibiting its ability to downregulate the NFkappaB-dependent gene expression. Thus, it can be expected that MC may have potential for therapeutic intervention on various NFkappaB-dependent pathological conditions such as inflammatory or possibly mutagenic processes.

The p73 gene located at 1p36.3 encodes for a protein with significant similarity to p53. To investigate the penetrance of p73 in gastric carcinogenesis, we analyzed the expression, allelotype, and mutation of p73 in five cell lines and 75 tissues. Although extremely low levels of p73 expression were observed in all noncancerous gastric tissues and four of five cell lines, a significant elevation of p73 was detected in 37 of 39 (94.9%) carcinoma tissues. Furthermore, a tumor-specific increase of p73 was identified in 14 of 16 (87.5%) matched sets. Allelotyping analysis using a StyI or BanI polymorphism revealed that 5 of 21 (23.8%) informative carcinomas, but none of 19 noncancerous cases, express p73 biallelically, suggesting the transcriptional activation of a silent allele in a subset of cancers. Whereas the transcription of an active allele was markedly induced by serum starvation or clump formation of the cells, treatment with 5-aza-2'deoxycytidine activated a silent allele with a subsequent up-regulation of an active allele, supporting the genomic imprinting and autoregulation of the gene. Allelic deletion or mutation of the gene was not found, and no association of p73 expression with the mutational status of p53 or expression of p21Waf1 was recognized. Taken together, this study argues that p73 is not a target of genetic alteration in gastric carcinogenesis and suggests that overexpression of p73 might be triggered by physiological stresses accompanied with outgrowth of tumors, such as hypoxia or nutrient deprivation.

Cyclins are overexpressed in various malignancies, including carcinoma of the colorectum, esophagus, lung, larynx, and breast. However, to the authors' knowledge, the protein levels and activities of cyclin-dependent kinases (CDKs), which are the functional cyclin partners in the cell cycle, have not been investigated previously.Eight samples of cancer tissue and adjacent normal tissue were taken from 23 patients with Stage B2-C1 (AJCC/UICC Stage II-III) colorectal carcinoma during curative resection. The protein levels of cyclin and CDKs were determined by Western blot analysis. The activities of CDKs were determined by the phosphorylation amount using specific substrates after immunoprecipitations.The protein expression of cyclin (D1, D3, E, and A) and CDKs (CDK4, CDK2, and cdc2) was higher in primary colorectal carcinoma tissue than in adjacent normal tissue. Whereas only 3 of 8 patients had increased CDK4 activity in cancer tissue, 8 of 8 and 7 of 8 patients had increased CDK2 and cdc2 activities, respectively, in cancer tissue compared with adjacent normal tissue. However, there were no positive correlations among the pathologic staging or differentiation status and the increased ratio of cyclin protein, CDK protein, or CDK activity.These results indicate that significant activation of S and M phases of the cell cycle occurs in primary colorectal carcinoma.

The molecular mechanisms that regulate the cardiomyocyte cell cycle and its terminal differentiation remain largely unknown. To determine which cyclins or cyclin dependent kinases (CDKs) are important for cardiomyocyte proliferation, we examined the expression of cyclins and CDKs during normal cardiac development. All cyclins and CDKs were highly expressed during embryonic cardiac development, then they decreased at different rates after birth. The mRNAs and proteins of cyclins A and B (G2 and M phase cyclins) were found in embryonic and neonatal hearts, but were not detected in young or adult hearts. In contrast, while the mRNAs of cyclins D1, D2, D3, and E (G1 and S phase cyclins) were observed during all stages of development, the proteins of cyclins D1, D3, and E were observed in hearts at the young growth stage, although the levels decreased differently. Reverse transcriptase-polymerase chain reaction (RT-PCR) using specific cyclin B and D3 primers revealed that cyclins B and D3 originated from cardiomyocytes and noncardiomyocytes. The CDKs (cdc2, CDK2, and CDK4) were highly expressed during embryonic cardiac development and maintained almost constant levels during neonatal periods. However, they were expressed at very low levels at the young and adult stages. The pattern of proliferating cell nuclear antigen (PCNA) expression during cardiac development was similar to the expression of CDKs. These findings suggest that all cyclins and CDKs are involved in the cardiac cell cycle, and that marked and rapid reduction of mitotic cyclins may be associated with the withdrawal of the cardiac cell cycle after birth.

Histone acetylation plays an important role in the chromatin structure prior to zygotic gene expression during early embryonic development. Successful animal clones indicate that differentiated somatic nuclei must be reprogrammed to some extent during pre-implantation development. However, the molecular mechanisms regarding epigenetic reprogramming of somatic nuclei in the early-stage embryos are poorly understood. To test this, the patterns of hyperacetylated histone H4 lysine 5 (AcH4K5) in the nuclear-transferred (NT) embryos were monitored, comparing in vitro fertilized (IVF) embryos and Trichostatin A (TSA)-NT embryos with TSA-treated cells. The intensity signals of AcH4K5 were observed in early-stage embryos and somatic cells (bovine ear skin fibroblasts composed of about 80% at G0/G1 stage) by immunofluorescence analysis with anti-AcH4K5 using image the analyzer system, SigmaScan-pro V5.01 (SPSS, Inc., Chicago, IL, USA). Our data were analyzed by analysis of variance (ANOVA) using an SAS package (SAS Institute, Inc., Cary, NC, USA). Somatic cells were exposed to TSA (1 μM for 60 h), a specific inhibitor of histone deacetylase (HDAC), to induce hyperacetylation prior to somatic cell nuclear transfer. Signal intensity for AcH4K5 in TSA-treated cells (n = 80) was significantly increased (P < 0.05), which was approximately double compared to that of normal cells (n = 80). In normal cells, histone H4 acetylation was profoundly reduced from the pro-metaphase to the early telophase and then reappeared at the late telophase. Acetylation signals of TSA-treated cells gradually increased to the early anaphase, abruptly decreased at the late anaphase and the early telophase, and recovered during late telophase. During early embryonic development (1 cell to 8 cell stage), NT embryos (n = 8) were hypoacetylated at the metaphase, whereas IVF (n = 10) and TSA-NT embryos (n = 8) were hyperacetylated. Our findings demonstrate that aberrant epigenetic reprogramming of histone modification occurs as early as the pronuclear stage in cloned embryos.

DNA demethylation as an epigenetic change is a unique event genome-wide, occurring at preimplantation and germ cell stages during mouse development. The paternal genome after fertilization is demethylated first, referred to active demethylation, followed by demethylation of the maternal genome during preimplantation development in the mouse. To examine methylation changes in the early mouse embryo, methylation states of various genomic regions such as intracisternal A-particle (IAP), early retrotransposon (Etn) and centromeric satellite sequences were determined by means of a bisulfite sequencing method. For methylation analysis, genomic DNA was first isolated from each developmental stage of embryo (about 300 cells in total), respectively, and exposed to 1.9M sodium bisulfite overnight. Targeted DNA sequences were amplified from bisulfite-treated genomic DNAs by PCR, cloned into pGEM T-easy vector and sequenced. Results indicated that IAP sequences maintained high levels of methylation until the morula stage and were demethylated in blastocysts. In contrast to the IAP sequences, methylation states of Etn elements were remarkably erased after fertilization, completely demethylated at the 8-cell stage and then remethylated at the morula stage. Centromeric satellite sequences showed low methylation states throughout all preimplantation stages of embryos, indicating that the satellite sequences are substantially demethylated in both paternal and maternal genomes. The results suggest that differential epifenetic changes among the repetitive sequences may be responsible for peculiar chromatin structure of respective genomic loci and/or may regulate gene expression during preimplantation development in the mouse.