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Background In a few studies, obesity was associated with better outcomes in patients with cardiogenic shock (CS). Although this phenomenon, the “obesity paradox”, reportedly manifests differently based on sex in other disease entities, it has not yet been investigated in CS patients. Methods and results 1,227 patients with CS from The REtrospective and prospective observational Study to investigate Clinical oUtcomes and Efficacy of left ventricular assist device for Korean patients with cardiogenic shock (RESCUE) registry in Korea were analyzed. The study population was classified into obese and non-obese groups according to Asian-Pacific criteria (BMI >25.0 kg/m2 for obese). Clinical impact of obesity on in-hospital mortality according to sex was analyzed using logistic regression analysis and restricted cubic spline curves. In-hospital mortality rate was significantly lower in obese men than non-obese men (34.2% vs. 24.1%, p=0.004) while the difference was not significant in women (37.3% vs. 35.8%, p=0.884). As a continuous variable, higher BMI showed a protective effect in men conversely, BMI was not associated with clinical outcomes in women. Comparing to normal-weight patients, obesity was associated with a decreased risk of in-hospital death in men (multivariable-adjusted OR 0.63, CI 0.43–0.92, p=0.016), not in women (multivariable-adjusted OR 0.94, 95% CI 0.55–1.61, p=0.828). Interaction P value for the association between BMI and sex was 0.023. Conclusions Obesity paradox exists and apparently occurs in men among CS patients. The differential effect of BMI on in-hospital mortality was observed according to sex. Funding Acknowledgement Type of funding sources: None.

Background Regarding stent expansion indexes, previous optical coherence tomography (OCT) studies have shown minimal stent area (MSA) to be most predictive of adverse events. Purpose We sought to evaluate the impact of various stent expansion indexes by post-stent OCT on long-term clinical outcomes, and hence to find OCT-defined optimal stent expansion criteria. Methods Of the patients registered in the Yonsei OCT registry, a total of 1071 patients with 1123 native coronary artery lesions treated with new-generation drug-eluting stents under the OCT guidance and analyzable final post-stent OCT were included. Stent expansion indexes and different suboptimal stent expansion criteria were evaluated for their association with device-oriented clinical endpoints (DoCE) including cardiac death, target vessel-related myocardial infarction (TVMI) or stent thrombosis, and target lesion revascularization. Major safety events (MSE) included cardiac death, TVMI or stent thrombosis. Results The median follow-up period was 40.6 (interquartile range 22.0–50.0) months. As a continuous variable, MSA, adaptive volumetric stent expansion (stent volume/adaptive reference lumen volume) and overall volumetric stent expansion (stent volume/post-stent lumen volume) were significantly predictive of DoCE. As a categorical criteria, MSA Conclusions This OCT study highlights that sufficient stent expansion to achieve adequate absolute MSA and relative MSA by distal reference lumen area and alleviate significant malapposition is important to improve clinical outcome, but overall stent overexpansion may have deleterious effect. Funding Acknowledgement Type of funding sources: None.

Background Even though the presence of hyperglycemia has shown to affect the clinical outcome of cardiogenic shock patients, the extent of hyperglycemia and its association with prognosis have not been fully addressed in large population Purpose Investigate the clinical relationship between hyperglycemic status and in-hospital mortality in cardiogenic shock patients Method A total of 1,177 consecutive cardiogenic shock patients were enrolled from January 2014 to December of 2018 at 12 hospitals in South Korea. The primary outcome was in-hospital mortality. Patients were divided into four groups according to their initial plasma glucose level in each of diabetes patients (n=752) and non-diabetes patients (n=425); group 1 (≤8 mmol/L), group 2 (8–12 mmol/L), group 3 (12–16 mmol/L) and group 4 (≥16 mmol/L). Results The groups with higher admission plasma glucose were associated with lower systolic blood pressure and higher lactic acid level in both diabetic and non-diabetic patients. In-hospital mortality increased in groups with higher admission plasma glucose level in non-diabetic patients (group-1:24.2%, group-2: 28.6%, group-3: 38.1%, group-4: 49.0%, p Conclusion In cardiogenic shock patients, plasma glucose obtained at admission was associated with in-hospital mortality in non-diabetic patients Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Inha University hospital

Vitrification is the most popular technique for the cryopreservation of oocytes and embryos, replacing slow freezing methods.This study evaluated the efficient manufacturing methods of handmade open pulled straw (OPS) with a digital heating gun that could be proposed for vitrification.Production efficiency of OPS using 0.5 mL straw was detected at 0, 66.1, 90.5 and 85.7% for 1~2 s and 9.5, 33.3, 47.6 and 23.8% for 2~3 s of heating time at 250, 350, 400 and 450°C respectively. The production rate of OPS using 0.25 mL straw was perceived at 33.3, 76.2, 83.3, 95.2 and 57.6 % for 1.5~2.5 s time with the optimized heat setting at 330, 340, 350, 360 and 370°C respectively. The desired inner diameter (200~300 μm) of OPS could be varied according to the gamete size, embryo developmental stages or cell lines of different species. Based on our data, the production efficiency of OPS using 0.25-mL straw were increased beyond using a 0.5-mL straw.Handmade OPSs could be efficiently produced with a digital heating gun to generate a vitrification device for freezing gametes, embryos and cell lines.

Background Machine learning approaches using intravascular optical coherence tomography (OCT) to predict fractional flow reserve (FFR) have not been previously investigated. The objective of the study was to evaluate a machine learning method to estimate FFR based on intravascular OCT images in intermediate coronary lesions. Methods Data from both OCT- and wire-based FFR methods were obtained for lesions of the left anterior descending artery in 125 patients. Based on the total number of lesions, training and testing groups were partitioned at a ratio of 5:1. For the training group, 36 features, including 16 clinical and lesion characteristics, and 21 OCT features, were used to model machine learning-FFR. machine learning-FFR values were then derived for the testing group and compared with wire-based FFR values in terms of a diagnosis of ischemia (FFR Results Clinical and lesion characteristics and OCT features between the training and testing groups were similar. During the machine learning modeling of the training group, six important features of machine learning-FFR were identified: minimal luminal area, percentage of the stenotic area, lesion length, proximal luminal area, pre-procedural platelet count, and hypertension. machine learning-FFR values showed a good correlation (r=0.853, P Conclusions The OCT-based machine learning-FFR method can be used to simultaneously acquire information on both image and functional modalities using one invasive procedure, suggesting that it may be used to optimize treatments for intermediate coronary artery stenosis, as well as save time and cost. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Funded by the Korean government (MSIT) (no. 2017R1A2B2003191)

MicroRNAs (miRs) are crucial regulators of vascular endothelial cell (EC) functions, including migration, proliferation, and survival. However, the role of most miRs in ECs remains unknown. Using RNA sequencing analysis, we found that miR-148a/b-3p expression was significantly downregulated during the differentiation of umbilical cord blood mononuclear cells into outgrowing ECs and that decreased miR-148a/b-3p levels were closely related to EC behavior. Overexpression of miR-148a/b-3p in ECs significantly reduced migration, filamentous actin remodeling, and angiogenic sprouting. Intriguingly, the effects of decreased miR-148a/b-3p levels were augmented by treatment with vascular endothelial growth factor (VEGF). Importantly, we found that miR-148a/b-3p directly regulated neuropilin-1 (NRP1) expression by binding to its 3′-untranslated region. In addition, because NRP1 is the coreceptor for VEGF receptor 2 (VEGFR2), overexpression of miR-148a/b-3p inhibited VEGF-induced activation of VEGFR2 and inhibited its downstream pathways, as indicated by changes to phosphorylated focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase. Collectively, our results demonstrate that miR-148a/b-3p is a direct transcriptional regulator of NRP1 that mediates antiangiogenic pathways. These data suggest that miR-148a/b-3p is a therapeutic candidate for overcoming EC dysfunction and angiogenic disorders, including ischemia, retinopathy, and tumor vascularization., Vascular biology: MicroRNA implicated in blood vessel formation A small regulatory RNA molecule helps prevent the development of new blood vessels, a finding that could have implications for the treatment of vascular disease and cancer. Young-Guen Kwon from Yonsei University in Seoul, South Korea, and colleagues cataloged all the microRNAs expressed during the differentiation of umbilical cord blood stem cells into precursors of the cells that line the inside of blood vessels. One microRNA in particular stood out for its association with cellular differentiation. The authors showed that this microRNA, called miR-148a/b-3p, directly binds part of the gene transcript encoding neuropilin-1, thereby blocking the production of this receptor for growth factors involved in new blood vessel formation. Modulating the activity of miR-148a/b-3p could have therapeutic value for disorders marked by aberrant blood vessel growth.

Background There is some debate on whether or not there is a gender difference is present between current smoking and cardiovascular disease. Purpose We decide to evaluate the impact of sex difference on the 2-year clinical outcomes in Korean acute myocardial infarction (AMI) patients who currently smoke and who underwent percutaneous coronary intervention (PCI) with drug-eluting stents (DES). Methods The data of this study was obtained from the Korea Acute Myocardial Infarction (KAMIR) registry. More than fifty high-volume universites or community hospitals with facilities for primary PCI and onsite cardiac surgery participated in this KAMIR registry. A total of 12565 current smoker AMI patients were enrolled and divided into the male (n=11767, 93.6%) or female (n=798, 6.4%) group. The clinical endpoint was the occurrence of major adverse cardiac events (MACE) defined as all-cause death, recurrent myocardial infarction (re-MI), and total repeat revascularization. Results Before risk adjustment, the cumulative incidences of MACE (7.2% vs. 10.0%, hazard ratio [HR], 1.419; 95% confidence interval [CI], 1.125–1.790; P=0.003), all-cause death (HR, 1.988; 95% CI, 1.417–2.789; P Clinical outcomes at 2-year Outcomes Cumulative Events at 2-year (%) Unadjusted Adjusted* Propensity-score adjusted Male Female Log-rank Hazard Ratio (95% CI) p value Hazard Ratio (95% CI) p value Hazard Ratio (95% CI) p value MACE 819 (7.2) 78 (10.0) 0.003 1.419 (1.125–1.790) 0.003 1.047 (0.756–1.450) 0.782 1.133 (0.871–1.473) 0.352 All-cause death 283 (2.5) 38 (4.8) Conclusion Before risk adjustment, a gender difference was suggested in the female group compared with the male in these current smoker South Korean AMI patients during a 2-year follow-up period. However, after adjustment, gender difference was not observed in these AMI patients with a history of current smoking.

Background There are limited comparative data concerning long-term major clinical outcomes between male and female in dyslipidemic AMI patients after PCI with new-generation DES. Purpose We thought to investigate the impact of gender difference on the 2-year clinical outcomes. Methods Finally, a total of 2403 eligible dyslipidemic AMI patients who underwent PCI with new-generation DES were enrolled and they were separated into two groups; the male group (n=1800) and the female group (n=603). The primary endpoint was major adverse cardiac events (MACE) defined as all-cause death, recurrent myocardial infarction (re-MI), target lesion revascularization (TLR), and target vessel revascularization (TVR). The secondary endpoints were the incidence of the individual components of MACE and target vessel failure (TVF), a composite of death related to the target vessel, re-MI, or clinically driven TVR Results Two propensity score-matched (PSM) groups (422 pairs, n=844, C-statistic = 0.850) were generated. In the total study population, the cumulative incidences of MACE, all-cause death, re-MI, and TVF were significantly higher in the female group compared with the male group. However, after propensity score-matched (PSM) analysis, the cumulative incidences of MACE (HR, 0.971; 95% CI, 0.628–1.501; p=0.895), all-cause death (HR, 1.061; 95% CI, 0.536–2.100; p=0.865), re-MI (HR, 1.212; 95% CI, 0.433–2.907; p=0.813), and TVF (HR, 0.764; 95% CI, 0.474–1.229; p=0.267) were similar between the two groups. In addition, the cumulative incidences of cardiac death, TLR, TVR were not significantly different between the two groups (Table 1). Outcomes Cumulative Events at 2-year (%) Hazard Ratio (95% CI) p value Male Female Log-rank Propensity score matched patients MACE 41 (10.4) 40 (10.3) 0.895 0.971 (0.628–1.501) 0.895 All-cause death 16 (4.0) 17 (4.2) 0.865 1.061 (0.536–2.100) 0.865 Cardiac death 13 (3.3) 9 (2.2) 0.391 0.691 (0.295–1.616) 0.393 Re-MI 8 (2.0) 9 (2.4) 0.813 1.212 (0.433–2.907) 0.813 TLR 7 (1.8) 6 (1.6) 0.781 0.857 (0.298–2.550) 0.781 TVR 16 (4.3) 10 (2.7) 0.236 0.623 (0.283–1.373) 0.241 TVF 39 (10.2) 30 (7.8) 0.265 0.764 (0.474–1.229) 0.267 Conclusion The gender difference was not apparent in these dyslipidemic South Korean AMI patients who underwent PCI with new-generation DES during 2-year follow-up period.

Introduction SPRINT showed that intensive control of systolic blood pressure (SBP) Methods SPRINT and ACCORD-BP databases were obtained via BioLINCC. Since BP changes along with the time, timing at which BP became stable after treatment were chosen at 9 months for SPRINT and 1 year for ACCORD BP. The final analytic datasets consisted of 8,787 and 4,306 event-free patients at the time of stable BP, respectively. Clinical events were defined at the primary composite endpoint for each trial. The effects of BP on clinical events were modeled using natural cubic splines in Cox regression models. Degrees of freedom of splines curves were chosen using Akaike Information Criterion. Results Analyzing SPRINT database, the spline-based hazard ratio (HR) curve showed a J-curve, which means lowering DBP below 77mmHg could be harmful, while SBP had a linear relationship (HR 1.13 per 10mmHg increase, 95% confidence interval [CI] 1.06∼1.20, p Conclusions Despite the different results regarding the benefit of intensive BP control, the effects of SBP on clinical events were quite similar in SPRINT and ACCORD-BP trial. Instead,differences existed in the relationships between DBP and clinical events. Understanding the meaning of different patterns of relationships including J-curve might be the key to determine the optimal BP control.

Background Although European guideline recommends that statin should be given to all patients with acute myocardial infarction (AMI), irrespective of cholesterol concentration, limited studies were focused on the long-term effects of statin therapy between ST-segment elevation MI (STEMI) and non-ST-segment elevation MI (NSTEMI). Purpose The authors conducted the study to compare the relative beneficial role of statin on 2-year major clinical outcomes between STEMI and NSTEMI in patients who underwent successful PCI with DES. Methods Finally, a total of 26317 AMI patients who underwent stent implantation and who were prescribed the statin were enrolled and they were separated into two groups; the STEMI group (n=15002) and the NSTEMI group (n=11315). The clinical endpoint was the occurrence of major adverse cardiac events (MACE) defined as all-cause death, recurrent myocardial infarction (re-MI), total coronary revascularization (target lesion revascularization [TLR], target vessel revascularization [TVR], non-TVR) during 2-year follow-up period. Results After propensity score-matched (PSM) analysis, two PSM groups (7746 pairs, n=15492, C-statistic = 0.766) were generated. In the total study population, the cumulative incidences of MACE, all-cause death, and cardiac death were significantly higher in the NSTEMI group. However, after PSM, the cumulative incidence of all-cause death (Hazard ratio, 1.386; 95% CI, 1.133–1.696; p=0.002) was significantly higher in the NSTEMI group. The cumulative incidences of MACE, cardiac death, re-MI, total revascularization, TLR, TVR, and non-TVR were similar between the two groups (Table 1). Outcomes Cumulative Events at 2-year (%) Hazard Ratio (95% CI) p value STEMI NSTEMI Log-rank Propensity score matched patients MACE 532 (7.2) 584 (8.1) 0.092 1.106 (0.984–1.244) 0.092 All-cause death 163 (2.2) 224 (3.1) 0.001 1.386 (1.133–1.696) 0.002 Cardiac death 121 (1.5) 148 (2.0) 0.088 1.232 (0.969–1.566) 0.089 Re-MI 117 (1.6) 107 (1.5) 0.545 0.922 (0.710–1.199) 0.545 Total revascularization 291 (4.1) 307 (4.4) 0.422 1.068 (0.910–1.254) 0.423 TLR 92 (1.3) 89 (1.2) 0.880 0.978 (0.731–1.309) 0.880 TVR 173 (2.4) 184 (2.6) 0.478 1.078 (0.876–1.327) 0.478 Non-TVR 123 (1.7) 130 (1.9) 0.593 1.070 (0.836–1.369) 0.539 Conclusion The mortality reduction capability of statin was more prominent in the STEMI group compared with the NSTEMI group.

Background Limited comparative data are available. Purpose We decided to compare 2-year major clinical outcomes between statin with ACEI and statin ARB therapy in patients with STEMI after PCI with drug-eluting stents (DES). Methods A total 11706 STEMI patients who underwent PCI with DES and who prescribed statin were enrolled and they were divided into two groups, the statin with ACEI group (n=8705) and the statin with ARB group (n=3001). The primary endpoint was the major adverse cardiac events (MACE) defined as all-cause death, recurrent myocardial infarction (re-MI), target lesion revascularization (TLR), target vessel revascularization (TVR), non-TVR. The secondary endpoints were the cumulative incidences of individual components of MACE and target vessel failure (TVF), a composite of death related to the target vessel, re-MI, or clinically driven TVR. Results Two PSM groups (2835 pairs, n=5670, C-statistic = 0.680) were generated. The relative risk of MACE was higher in the statin with ARB group compared to statin with ACEI groups after propensity score-matched (PSM) analysis (hazard ratio [HR]: 1.323, 95% confidence interval [CI]: 1.085–1.613, p=0.006). The relative risks of cardiac death (HR: 1.831, 95% CI: 1.199–2.740, p=0.005), total repeat revascularization (HR: 1.487, 95% CI: 1.133–1.950, p=0.004), and non-TVR (HR: 1.696, 95% CI: 1.122–2.564, p=0.012) were also higher in the statin with ARB group after PSM. Outcomes Cumulative Events at 2-year (%) Hazard Ratio (95% CI) p value Statin + ACEI Statin + ARB Log-rank Propensity score matched Patients MACE 173 (6.5) 225 (8.5) 0.006 1.323 (1.085–1.613) 0.006 All-cause death 58 (2.2) 80 (3.0) 0.054 1.391 (0.992–1.950) 0.056 Cardiac death 35 (1.3) 63 (2.3) 0.004 1.831 (1.199–2.740) 0.005 Re-MI 39 (1.5) 44 (1.7) 0.548 1.141 (0.742–1.756) 0.548 Total repeat revascularization 88 (3.4) 128 (4.9) 0.004 1.487 (1.133–1.950) 0.004 TLR 26 (1.0) 40 (1.5) 0.075 1.561 (0.953–2.558) 0.077 TVR 53 (2.0) 71 (2.8) 0.086 1.364 (0.955–1.946) 0.088 Non-TVR 36 (1.4) 60 (2.3) 0.011 1.696 (1.122–2.564) 0.012 TVF 140 (5.3) 173 (6.6) 0.050 1.249 (1.000–1.561) 0.050 Conclusions In this study, we suggest that the combination of statin with ACEI may be beneficial for reducing the cumulative incidences of MACE, total repeat revascularization rate, and non-TVR rather than the statin with ARB after PCI with DES in STEMI patients.

The successful cryopreservation of spermatozoa of the beagle dog for AI is essential for the establishment of the genetic banks of drug detection dogs. The beagle dog is widely used for drug testing and chosen for breeding by breeders. However, the use of cryopreserved beagle semen is limited by the lower number of offspring of dog species. In this study, 3 highly trained beagle dogs were chosen and their semen was cryopreserved for the next generation. The effects of dilution methods of beagle semen were tested using a direct dilution method at RT and a 2-step dilution method at 5°C. As a control group, the effects of a direct dilution method of semen on the percentage of motile sperm and progressive motility were analysed by computer-assisted semen analysis system (SAIS, Korea), and abnormality of spermatozoa was examined by Diff Quik staining. A total of 9 samples from 3 dogs were extended in 4% glycerol containing Tris-egg yolk diluents at approximately 22 to 25°C. The diluted semen was cooled to 5°C within 2h. The packed 0.5-mL straws were placed 5cm above the surface of LN for 10min and then plunged in. A 2-step dilution method was conducted using the same procedures of freezing, but the first dilution was done with glycerol-free diluent. After cooling to 5°C within 2h, the second diluent with 8% glycerol was added to the same volume of diluted semen at 5°C and stabilised for 1h. After thawing for 45s at 37°C, the semen from the 2-step dilution method showed the higher percentage of motile sperm (65.4±6% v. 45.3±8%; P

The influence of the cloned-cattle meat diets upon reproduction in mammals was rarely studied. This study was performed to analyze the effects of the diets containing cloned-cattle (Korean native beef, Hanwoo) meat on the reproductive physiology in rats. The male and female rats were fed with the diets containing 5% or 10% of normal- (N-5 or N-10) or cloned- (C-5 or C-10) cattle meat during test periods. The rats fed with commercial pellets were used as control. Lower food consumption in normaland cloned-cattle meat diet groups is detected in both male and female rats compared with that of control ( P , 0.05, 0.01 and 0.001). No signs of cloned-cattle meat diets on male reproductive parameters are found in all groups, except for lower sperm deformity in C-5 group ( P , 0.05) and higher testosterone concentration in C-10 group ( P , 0.05), respectively. There are no significant test substance-related differences of Caesarean section and delivery in dams and external examination and physiological development test in neonate compared with control and normal meat groups. Based on these results, it can be postulated that there are no obvious negative effects on the reproductive physiology in rats fed with cloned-cattle meat diets compared to their comparators.

Objective: To evaluate the vascular response at 9 months after zotarolimus-eluting stent (ZES; Endeavor) implantation using optical coherence tomography (OCT). These findings were compared with those after implantation of a sirolimus-eluting stent (SES; Cypher Select). Design: Cross-sectional observational study with prospective OCT registry. Setting: Nine months after ZES or SES implantation. Patients and methods: A total of 68 patients (32 ZES and 36 SES) underwent OCT at 9 months after stent implantation. The neointima hyperplasia (NIH) thickness inside each strut and percentage of NIH area at every 1 mm cross section were measured. Main outcome measurement: The degree of neointimal coverage and the prevalence of malapposition at 9 months after ZES and SES implantation using OCT. Results: The mean (SD) NIH thickness (251.2 (110.0) μm vs 85.5 (53.3) μm, p

AMIGO2 is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation in endothelial cells, and inhibition of the interaction between PDK1–AMIGO2 results in impaired neovascularization, pathological angiogenesis, and tumor angiogenesis., The phosphoinositide 3-kinase–Akt signaling pathway is essential to many biological processes, including cell proliferation, survival, metabolism, and angiogenesis, under pathophysiological conditions. Although 3-phosphoinositide–dependent kinase 1 (PDK1) is a primary activator of Akt at the plasma membrane, the optimal activation mechanism remains unclear. We report that adhesion molecule with IgG-like domain 2 (AMIGO2) is a novel scaffold protein that regulates PDK1 membrane localization and Akt activation. Loss of AMIGO2 in endothelial cells (ECs) led to apoptosis and inhibition of angiogenesis with Akt inactivation. Amino acid residues 465–474 in AMIGO2 directly bind to the PDK1 pleckstrin homology domain. A synthetic peptide containing the AMIGO2 465–474 residues abrogated the AMIGO2–PDK1 interaction and Akt activation. Moreover, it effectively suppressed pathological angiogenesis in murine tumor and oxygen-induced retinopathy models. These results demonstrate that AMIGO2 is an important regulator of the PDK1–Akt pathway in ECs and suggest that interference of the PDK1–AMIGO2 interaction might be a novel pharmaceutical target for designing an Akt pathway inhibitor.

Item does not contain fulltext BACKGROUND: The effect of intensified platelet inhibition for patients with unstable angina or myocardial infarction without ST-segment elevation who do not undergo revascularization has not been delineated. METHODS: In this double-blind, randomized trial, in a primary analysis involving 7243 patients under the age of 75 years receiving aspirin, we evaluated up to 30 months of treatment with prasugrel (10 mg daily) versus clopidogrel (75 mg daily). In a secondary analysis involving 2083 patients 75 years of age or older, we evaluated 5 mg of prasugrel versus 75 mg of clopidogrel. RESULTS: At a median follow-up of 17 months, the primary end point of death from cardiovascular causes, myocardial infarction, or stroke among patients under the age of 75 years occurred in 13.9% of the prasugrel group and 16.0% of the clopidogrel group (hazard ratio in the prasugrel group, 0.91; 95% confidence interval [CI], 0.79 to 1.05; P=0.21). Similar results were observed in the overall population. The prespecified analysis of multiple recurrent ischemic events (all components of the primary end point) suggested a lower risk for prasugrel among patients under the age of 75 years (hazard ratio, 0.85; 95% CI, 0.72 to 1.00; P=0.04). Rates of severe and intracranial bleeding were similar in the two groups in all age groups. There was no significant between-group difference in the frequency of nonhemorrhagic serious adverse events, except for a higher frequency of heart failure in the clopidogrel group. CONCLUSIONS: Among patients with unstable angina or myocardial infarction without ST-segment elevation, prasugrel did not significantly reduce the frequency of the primary end point, as compared with clopidogrel, and similar risks of bleeding were observed. (Funded by Eli Lilly and Daiichi Sankyo; TRILOGY ACS ClinicalTrials.gov number, NCT00699998.).

To understand the tissue-specific expression of the rat placental lactogen-I variant (rPL-Iv) gene, we investigated the methylation pattern of the 5'-flanking region of this gene in various rat tissues. We report that the 5'-flanking region of the rPL-Iv gene was hypomethylated in placenta that expressed the gene and hypermethylated in those tissues that did not express the gene. Moreover, the intron region of the rPL-Iv gene was hypomethylated in the placenta, but hypermethylated in the liver, kidney and pituitary. Although there are 5 CpG sites and the density of CpG dinucleotide is lower within 2 kb of the rPL-Iv 5'-flanking region, the methylated promoter reporter gene produced strong repression in the transcriptional activity of the gene. In addition, the 5'-flanking and intron regions of the rPL-Iv gene were hypomethylated on day 12 of gestation, and the methylation pattern in the placenta remained unchanged from mid-pregnancy until term. The entire genomic region of the rPL-Iv gene might be hypermethylated in tissues other than the placenta, within which its methylated status repress expression of the placenta-specific rPL-Iv gene. Interestingly, the methylation status of the intron region of the rPL-Iv in proliferating Rcho-1 cells was changed to the unmethylated status on day 8 and 12 of differentiation of Rcho-1 cells. These results demonstrate that demethylation in the rPL-Iv upstream region was induced at an early stage of placental development, and once the 5'-flanking region of the rPL-Iv had been demethylated, its status on the rPL-Iv genomic region was continued during pregnancy. Taken together, these results suggest that DNA methylation is responsible for the silencing of tissue-specific genes in non-expressing cells, while defined combinations of trophoblast factors dictate the expression of unmethylated rPL-Iv gene in placenta trophoblast cells.

FADD is a mammalian pro-apoptotic mediator consisting of the N-terminal death effector domain (DED) and the C-terminal death domain (DD). The N-terminal 88-residue fragment of murine FADD was defined as the stable structural unit of DED, as determined by proteolytic digestion and conformational analysis. This domain induced bacterial as well as mammalian cell death, whereas the full-length or DD of FADD did not. The Escherichia coli cells expressing FADD-DED showed elongated cell morphology and an increased level of nicked chromosomal DNA and mutation. The lethality of FADD-DED was abolished by co-expression of thioredoxin and superoxide dismutase or relieved by the addition of vitamin E as a reducing agent and under anaerobic growth conditions. The toxicity of FADD-DED was genetically suppressed by various oxidoreductases of E. coli. All these results suggest that the death effector domain of mammalian FADD induced bacterial cell death by enhancing cellular levels of reactive oxygen species (ROS).

Caveolae-like domains (CLDs) have been hypothesized to mediate apoptosis, since they contain sphingomyelin and initiate the conversion of sphingomyelin to ceramide. To address whether CLDs are directly involved in apoptosis, CLDs from U937 cells were isolated, taking advantage of their detergent insolubility and low density. The CLDs contained alkaline phosphatase as well as many signaling molecules, including Fyn, protein kinase Calpha, Raf-1, phospholipase Cgamma1, and tyrosine phosphoproteins. Immunoblotting and immunofluorescent data showed that TNF receptor 1 colocalized with CD36 in CLDs, suggesting that TNF-alpha-initiated apoptosis occurs in CLDs. When cells were incubated with lipoprotein-deficient medium, the cholesterol concentration was greatly decreased in CLDs but not in other fractions, implying that the CLDs were selectively disrupted. In the CLD-disrupted cells, the surface expression of TNF receptor 1 and CD36 was significantly reduced. Analysis of cellular morphology, percent DNA fragmentation, DNA laddering, and caspase-3 activity showed that TNF-alpha-mediated apoptosis was blocked in CLD-disrupted cells, whereas anti-Fas-mediated apoptosis was not. Since Fas was not found in CLDs of Jurkat cells, apoptosis by Fas ligation might not require CLDs. Taken together, these data strongly imply that TNF-alpha-mediated apoptosis is initiated in CLDs.

Ceramide has been known as an important second messenger in programmed cell death (apoptosis) which is induced by various stimuli such as the tumor necrosis factor-alpha (TNF-alpha), Fas ligand, and environmental stresses such as UV-irradiation and heat shock. Although the precise molecular mechanism of apoptosis is not fully understood, ceramide generated by sphingomyelinase (SMase) mediates the activation of several downstream molecules that are implicated in the regulation of apoptosis. Here, we show that stress-inducible heat shock protein 70 (Hsp70) prevents apoptosis induced by increased level of intracellular ceramide. In T-cell hybridoma DO11.10, we examined the effect of Hsp70 on apoptosis mediated by TNF-alpha, Fas ligation, SMase, and C2-ceramide, all of which elevate intracellular ceramide levels. Hsp70 not only markedly reduced internucleosomal DNA fragmentation, but also enhanced cell viability measured by the Trypan blue dye exclusion test. Similarly, the ceramide-induced c-jun amino-terminal kinase (JNK/SAPK) activation is impaired in cells overexpressing Hsp70. These data strongly suggest that hsp70 functions as a regulator of apoptosis downstream of ceramide.

Members of the nonreceptor tyrosine kinase family appear to be targeted to caveolae membrane. We have used a Rat-1 cell expressing a temperature sensitive pp60(v-src) kinase to assess the initial changes that take place in caveolae after kinase activation. Within 24-48 h after cells were shifted to the permissive temperature, a set of caveolae-specific proteins became phosphorylated on tyrosine. During this period there was a decline in the caveolae marker protein, caveolin-1, a loss of invaginated caveolae, and a 70% decline in the sphingomyelin content of the cell. One of the phosphorylated proteins was caveolin-1 but it was associated in coimmunoprecipitation assays with both a 30 kDa and a 27 kDa tyrosine-phosphorylated protein. Finally, the cells changed from having a typical fibroblast morphology to a rounded shape lacking polarity. In light of the recent evidence that diverse signaling events originate from caveolae, pp60(v-src) kinase appears to cause global changes to this membrane domain that might directly contribute to the transformed phenotype.

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus, conservation of genetic material in chickens was attempted by preserving primordial germ cells (PGC) in LN2. This study established a method for preserving chicken PGC that enables long-term storage in LN2 for preservation of species. The purpose of this study was to clarify the effects of fetal bovine serum (FBS) or chicken serum (CS) treatment on the viability of cryopreserved PGC in the Korean native chicken (Ogye). Primordial germ cells were separated from a germinal gonad using a fine glass micropipette under a microscope and were suspended in a freezing medium containing freezing and protecting agents [e.g. dimethyl sulfoxide (DMSO) and ethylene glycol (EG)]. The PGC were then purified using the magnetic-activated cell sorting (MACS) method. The viability of the PGC in both groups was determined by the trypan blue exclusion method. The values of the 0, 5, 10, and 15% DMSO plus FBS treatment were 21.6, 30.36, 36.42, 50.39, and 48.36%, respectively. The viability of PGC after freeze-thawing was significantly higher for the 10% EG plus FBS treatment than for the 10% EG plus CS treatment (P

The purpose of this study was to evaluate the viability, acrosome integrity, mitochondrial activity, and fertility of frozen-thawed fowl semen using different cryoprotective agents. The experiments were carried out on 10 sexually adult roosters of the Ogye breed (Korean native black chicken). The semen was collected twice per week and diluted in a 1 : 1 ratio with EK extender without cryoprotectant at 5°C. After equilibration for 30 min, diluted semen was added with an equal volume of diluents containing 14% dimethylacetamide (DMA), 14% dimethylformamide (DMF), or 15% methylacetamide (MA). The semen were packed into 0.5-mL plastic straws, frozen for 30 min by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, and then plunged into liquid nitrogen at –196°C. Frozen semen was thawed in a cold water bath at 5°C for 2 min. For fluorescence-assisted cell sorting (FACS) analysis, the frozen-thawed semen was adjusted to a final concentration of 90 million spermatozoa per milliliter with EK extender. Sperm membrane integrity was evaluated by the live-dead staining method with SYBR-14 dye and propidium iodide (PI). Acrosome integrity was measured with fluorescein isothiocyanate-labelled Pisum sativum agglutinin (PSA) and PI. The percentage of functional mitochondria was estimated using Rhodamine 123 (R123) dye and PI. After intravaginal AI was performed twice per week by injecting 0.2 mL of thawed semen directly into the vagina within 2 min after thawing, the resulting eggs were incubated for 7 days to confirm fertilization. The survival rates of cryopreserved sperm with DMF, DMA, and MA were 52.1 ± 5.5, 46.9 ± 5.1, and 36.6 ± 4.7%, respectively. The survival rate of DMF was significantly higher than those of DMA and MA (P

This study was performed to increase the developmental rate of cloned embryos with the 1,3-Galactosyltransferase (GalT) gene knocked out (KO). Ovaries were collected from local slaughterhouse and immature oocytes were cultured in TCM-199 + 0.1% PVA + FSH + LH (0.5 μg mL-1) + EGF (10 ng mL-1) + 10% porcine follicular fluid (pFF) at 38.5°C in 5% CO2 humidified chamber for 40 h (1-step) or 20 h (with hormone) +20 h (without hormone; 2-step). After IVM, the oocytes with 1st polar body were enucleated and transferred the GalT KO donor cell originated from miniature pig. The embryos transferred with normal mini-pig ear fibroblast cell were used as control. The reconstructed embryos were fused with 2 electric pulses (DC) of 1.2 kV cm-1 for 30 μs. For the development of cloned embryos, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5°C for 6 days. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle, and pregnancy diagnosis was determined at 28 days after embryo transfer using ultrasonography. Differences among treatment means were determined by a chi-square test. A probability of P < 0.05 was considered statistically significant. The maturation rate was significantly higher in the 2-step method (89.8 ± 2.75) compared with single maturation method (79.6 ± 8.95; P < 0.05). The blastocyst development of cloned embryos reconstructed with GalT KO donor cell (28.4 ± 2.14) was not different from cloned embryos by normal donor cell (27.4 ± 0.01). The cell number of GalT blastocyst (36.1 ± 11.1) was not different statistically from control (26.9 ± 9.3). The apoptosis rate was also not different in both groups (2.9 to 4.9%). Five surrogates were pregnant and the GalT KO fetuses were still ongoing pregnancy at 45 days after embryo transfer. This work received grant support from the Agenda Program (No. 200901FHT010305146 and No. 200901FHT010305535), Rural Development Administration, Republic of Korea.

Instead of surgical embryo transfer (ET) in the pig, nonsurgical ET is a hopeful method to increase the efficiency of biotechnology applications such as cloning and transgenesis. In this study, we conducted surgical and nonsurgical ET methods after somatic cell nuclear transfer (SCNT) with MHC miniature pig cells to find out the best condition for production of cloned miniature pigs. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pigs. Fibroblast cells were cultured, passaged (3 to 8 passages), and used as donor cells for NT. After the enucleation and injection process, eggs were held in TCM-199. For fusion, 2 DC pulses of 1.2 kV cm-1 were applied for 30 μs. Both IVF and SCNT embryos were cultured in PZM-3 medium. After IVF, 84.9% (411/484) of embryos cleaved and 27.3% (132/484) of embryos reached the blastocyst stage. In the SCNT group, 80.8% (231/286) of eggs fused and 25.9% (60/286) of embryos developed to blastocysts. For surgical ET, approximately 200 SCNT embryos were transferred into oviducts of each synchronized recipient. For nonsurgical ET, embryos were cultured in PZM-3 for 6 days after SCNT and IVF, and then good quality blastocyst stage embryos were selected for ET. The pregnancy status of recipients at Day 30 was determined by ultrasound scanning. Using Day 30 of gestation as an endpoint, the nonsurgical ET method (47.3%, 9/19) had a similar pregnancy rate as the surgical ET method (56.5%, 13/23). Further study is needed to optimize the nonsurgical ET method especially for SCNT eggs. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.

Porcine cloning by somatic cell nuclear transfer (SCNT) has limitations because of the high incidence of fetal failure after embryo transfer to recipient. The fetal failure may originate from disturbed embryo-uterine interactions in the early implantation period. In this study, we compared apoptotic-related gene expression and cytokines using real-time RT-PCR and protein chip array. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pig. After the enucleation and injection process, eggs were held in TCM-199. For fusion, two DC pulses of 1.2 kV cm-1 were applied for 30 μs. SCNT embryos were cultured under PZM-3 medium to the 1- to 4-cell stage. For embryo transfer, approximately 200 SCNT eggs were transferred into the oviduct of each synchronized recipients. We obtained the extraembryonic (embryo origin) and endometrium (maternal origin) tissues from 3 cloned fetus with that from normal fetus by natural mating at Day 30. Each experiment was repeated at least 3 times and the data were presented as mean ± SE. Values of P < 0.05 were considered statistically significant. Data from real-time RT-PCR are expressed as log (fold change) ± SE. Expression of Bax-α, p53, and caspase-3 mRNA was significantly higher in the SCNT group than in the control group (P < 0.05). Five cytokines were analyzed: Interleukin (IL)4, IL8, tumor necrosis factor-α, and epidermal growth factor were lower in the control group than in the cloned group (P < 0.05). However, the level of MCP1 was higher in the SCNT group (P < 0.05). Numbers of trophoblast cells in the cloned group were also lower than in the control group (P < 0.05). These findings indicate that failure of endometrium and extraembryonic tissues in cloned pregnancies may originate from abnormal embryo-maternal communication that develops during the implantation period. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.

The postural stability of four adult populations was examined through force platform methods. The four groups were classified as: (1) developmentally disabled (severely and profoundly mentally retarded) with tardive dyskinesia; (2) developmentally disabled but with no history of neuroleptic medication; (3) tardive dyskinetic but of normal intelligence; and (4) a normal and healthy control group. Postural conditions included standing still with arms at side, standing still with one arm or both arms parallel to the ground, and standing still while swinging both arms in the sagittal plane. The findings showed that both tardive dyskinetic and/or developmentally disabled groups exhibited greater sway and variability in centre of pressure motion in contrast to the control group. The developmentally disabled with tardive dyskinesia group also exhibited a strong tendency to produce a different form to the postural sway strategy in that they produced rhythmical centre of pressure motions during stance that were, to some degree, task dependent. The findings show that the combined effects of developmental disability and tardive dyskinesia produce qualitatively and quantitatively different features in postural stability patterns. The data suggest that postural stability measures may be a useful index to assess tardive dyskinesia.

Manipulations of early embryos require that the embryos be placed in vitro. The ability to reproduce in vivo conditions in vitro would greatly facilitate studies on the development of early embryos. A variety of different conditions have been described that result in development of pig embryos from the 1-cell stage to the blastocyst stage in vitro. There is a species-specific cell stage at which the early embryo is very sensitive to in vitro conditions, which generally corresponds to the stage at which the embryo begins producing significant amounts of RNA. The present study was conducted to investigate the relative amounts of apoptotic gene expression in miniature pig NT embryos under culture conditions of different osmolarity. Oocytes were cultured in TCM-199 for 40–44 h at 38.5�C under 5% CO2 in air. Miniature pig ear fibroblast cells were cultured to reach confluency, and the culture was continued for an additional 5–6 days. The NaCl group of embryos was cultured in PZM-3 supplemented with 138 mM NaCl in total concentration (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for a further 4 days. The control group of embryos was cultured in the PZM-3 for the entire period of in vitro culture. Total RNA samples were prepared from 2 blastocysts using the Roche 1st strand cDNA synthesis kit. Bax and Bcl-xl gene expression of blastocysts was analyzed by real-time RT-PCR. Developemntal rates were analyzed by a GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Relative gene expression was compared by Student's t-test. Blastocyst formation rate in the NaCl group was not different from that in the control group (25.4% and 23.2%, respectively), but the apoptosis rate was significantly lower (P < 0.05) in the NaCl group (1.6%) than in the control (7.1%). The relative abundance of Bax mRNA expression was significantly higher (P < 0.05) in the control group (n = 32) than in the NaCl group (n = 33). However, the relative abundance of Bcl-xl mRNA was significantly higher (P < 0.05) in NaCl group. The relative abundance of Bax/Bcl-xl was significantly higher in the control group than in the NaCl group (P < 0.05). These results indicate that the hypertonic culture condition at the early embryonic stage of miniature pig NT embryos could reduce the frequency of apoptosis through regulating Bax and Bcl-xl gene expression.

In vitro production of the pig embryo is very important as an initial step to improve its application in biotechnology. The in vitro production system for pig embryos, however, has been plagued by the high incidence of polyspermy and poor embryo quality. The present study was conducted to examine the relationship between apoptosis and osmolarity of culture medium in pre-implantation development of porcine NT and IVF embryos. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 40–44 h. Fresh semen was diluted and equilibrated at 16�C. The final concentration of motile spermatozoa was adjusted to 5 � 105 cells/mL in fertilization medium. Fetal fibroblasts were prepared from a 35-day-old porcine fetus for use as donor cells. The NT and IVF embryos were cultured in PZM-3 supplemented with 0.05 M sucrose or a final concentration of 138 mM NaCl (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for the remaining days. For the control, NT and IVF embryos were cultured in PZM-3 for whole culture period. After 6 days of culture, the developmental ability of embryos, total cell numbers, ratio of ICM/TE, and apoptosis of cells in blastocysts were examined. The developmental rate to the blastocyst stage of NT embryos was significantly higher (P < 0.05) in the sucrose and NaCl groups than in the control [14.7% (21/153) and 21.7% (34/154) vs. 11.5% (18/152), respectively]. Also, the developmental rate to the blastocyst stage after IVF was slightly higher in embryos cultured in the medium supplemented with NaCl than in the control group [21.8% (49/235) and 26.4% (61/237) vs. 18.9% (44/247)]. For apoptosis, both NT and IVF blastocysts produced in the sucrose and NaCl groups showed slightly lower frequency of apoptosis compared to that of the control (2.2% and 2.8% vs. 3.1% for NT; 0.9% and 0.7% vs. 1.1% for IVF). These studies suggest that the high osmolarity in the early embryo culture stage could enhance the in vitro development of both porcine NT and IVF embryos to the blastocyst stage and could reduce the apoptosis of cells.

Telomeres are specialized nucleoprotein complexes at the termini of linear chromosomes that are composed of TTAGGG sequences in vertebrates. Telomere lengths in animals vary with species, age, tissue types, environment, and cloning. The experiment conducted emphasized the amount of telomeric DNA in the lymphocytes and organs of growing cloned cattle and their second and third generations. Using somatic cell nuclear transfer (SCNT), 16 cloned (Generation, clone G1) Korean Native cows were obtained from ear skin fibroblasts and 2 cloned bulls from fetal fibroblasts. In addition, 3 females and 2 males (clone G2) were produced from each cloned cow by artificial insemination (AI). A third generation calf (clone G3) was derived from clone G2 by AI. The lymphocytes of all cloned cattle (G1), their offspring (G2), and age-matched controls were examined 3 times at 6-month intervals whereas G3 was examined only once. The amount of telomeric DNA was analyzed by quantitative fluorescence in situ hybridization (Q-FISH) with a human telomeric DNA repeat probe. A minimum of 100 interphase nuclei from each set of harvests was studied to determine the mean and medium percentages of telomeric DNA using MetaMorph Imaging System (Universal Imaging Co, West Chester, PA, USA). The amounts of telomeric DNA in cloned cattle from both ear skin fibroblasts (female, n = 16) and fetal fibroblasts (male, n = 2) were less than those of age-matched controls (P < 0.01). Additionally, irrespective of gender, the telomeres in the clone G2 and G3 calves were lower than in controls (n = 6; P < 0.05). Furthermore, in the cloned cattle, the amount of telomeric DNA was drastically less than that of control animals during growth. Moreover, we examined the internal organs and tissues of a cloned cow at 30 months. The telomeres of leukocytes, cerebrum, spleen, cerebellum, hindbrain, and lung were a little smaller, whereas those of the liver, pituitary, kidney, and heart were slightly larger, than those of an age-matched cow. The results showed a remarkable difference in the amount of telomeric DNA between SCNT cloned cattle and normal cattle. Although the organs and tissues were not correlated, the amount of telomeres rapidly decreased with growth in cloned cattle. Conclusively, the telomeres of a cloned animal and its calves were significantly shorter than those of control cattle, and the short telomeres in calves could be inherited by progeny from their cloned mother.

Somatic cell nuclear transfer in pig has limitations due to the high incidence of fetal failure after embryo transfer to recipients. Reasons for the inefficient cloning are assumed to be due to abnormal and poorly developed placenta. Thus, this study was designed to determine possible genetic causes of neonatal deaths and other related abnormalities. Genes expressed specifically or prominently on Day 35 were identified in cloned pig placenta utilizing PCR technology regulated by annealing control primers (ACPs). The RNA was isolated using Trizol reagent. By utilizing 120 ACPs, 53 expressed sequence tags (ESTs) of genes that are differentially expressed in cloned pig placenta compared with normal placenta were cloned and sequenced. The cloned genes or ESTs exhibited significant sequence similarities to known genes or ESTs of other species. Ten of the total known genes, i.e., pregnancy associated glycoprotein, H19, 60S ribosomal protein L12, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, heme oxygenase 2, granulin precursor, and placenta-expressed transcript protein, were selected and their specific expression levels were confirmed by real-time RT-PCR in the normal and cloned pig placentas in triplicate using beta-actin for determining relative expression. The 60S ribosomal protein L12 and heme oxygenase 2 were highly expressed in the cloned pig placenta, whereas pregnancy associated glycoprotein, H19, 20-beta hydroxysteroid dehydrogenase, beta galactosidase precursor, aldehyde reductase, glypican 3 precursor, granulin precursor, and placenta-expressed transcript protein were low or void. Our data suggest that the ACP system effectively identified tissue-specific genes in cloned pig placenta. Furthermore, identified genes would assist in developing insight into the genetic basis of fetal failure and help in resolving low pregnancy rate in the production of cloned pigs.

A telomere is a structure consisting of tandem repeats sequences of (TTAGGG)n at the end of the eukaryotic chromosome. Telomere lengths in animals vary by species, age, and tissues, as well as environment. This experiment concentrated on the amount of telomeric DNA in cloned cattle, their calves, and age-matched normal cattle. Using somatic cell nuclear transfer (SCNT), we had obtained 16 cloned Korean Native cows derived from ear skin fibroblasts and two cloned bulls from fetal fibroblasts. In addition, four female calves were produced from each cloned cow by artificial insemination. Control cattle selected to have matched age and the same raising place served as counter-parts of cloned the cattle in this study. The lymphocytes of all cloned cattle, their calves, and the age-matched controls were examined for telomere quantity. The amount of telomeric DNA was analyzed by quantitative fluorescence after in situ hybridization (Q-FISH) with a human telomeric DNA repeat probe. A minimum of 100 interphase nuclei from each set of harvests was studied to determine the mean and medium percentages of telomeric DNA using the MetaMorph Imaging System (Universal Imaging Co., West Chester, PA, USA). The amount of telomeric DNA obtained was found to decrease in cloned and control animals during growth. The amounts of telomeric DNA in cloned cattle from both ear skin fibroblasts (n = 16) and fetal fibroblasts (n = 2) was less than that of age-matched controls (P < 0.01). Surprisingly, the amount of telomeric DNA of calves from cloned cattle was also lower than that of age matched controls (n = 4, P < 0.01). The results showed a remarkable difference in the amount of telomeric DNA between SCNT cloned cattle and normal cattle. In conclusion, the telomeres of cloned animal and their calves are significantly shorter than those of normal cattle. Moreover, the short telomeres in calves could be inherited from their cloned mothers.