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Characterization of MACS Isolated Cells from Differentiated Human ES Cells (인간 배아줄기세포로부터 분화된 세포에서 MACS 방법을 이용하여 분리한 세포의 특성에 대한 연구)

  • Cho, Jae Won;Lim, Chun Kyu;Shin, Mi Ra;Bang, Kyoung Hee;Koong, Mi Kyoung;Jun, Jin Hyun
    • Clinical and Experimental Reproductive Medicine
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    • v.33 no.3
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    • pp.171-178
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    • 2006
  • Objective: Human embryonic stem (ES) cells have a great potential in regenerative medicine and tissue engineering. The human ES cells could be differentiated into specific cell types by treatments of growth factors and alterations of gene expressions. However, the efficacy of guided differentiation and isolation of specific cells are still low. In this study, we characterized isolated cells from differentiated human ES cells by magnetic activated cell sorting (MACS) system using specific antibodies to cell surface markers. Methods: The undifferentiated hES cells (Miz-hESC4) were sub-cultured by mechanical isolation of colonies and embryoid bodies were spontaneously differentiated with DMEM containing 10% FBS for 2 weeks. The differentiated cells were isolated to positive and negative cells with MACS system using CD34, human epithelial antigen (HEA) and human fibroblast (HFB) antibodies, respectively. Observation of morphological changes and analysis of marker genes expression were performed during further culture of MACS isolated cells for 4 weeks. Results: Morphology of the CD34 positive cells was firstly round, and then it was changed to small polygonal shape after further culture. The HEA positive cells showed large polygonal, and the HFB positive spindle shape. In RT-PCR analysis of marker genes, the CD34 and HFB positive cells expressed endodermal and mesodermal genes, and HEA positive cells expressed ectodermal genes such as NESTIN and NF68KD. The marker genes expression pattern of CD34 positive cells changed during the extension of culture time. Conclusion: Our results showed the possibility of successful isolation of specific cells by MACS system from undirected differentiated human ES cells. Thus, MACS system and marker antibodies for specific cell types might be useful for guided differentiation and isolation of specific cells from human ES cells.

Improvement of Small Wetland and Stream in Cultivated Area in point of Landscape Ecology (경작지 내 소규모 습지 및 소하천의 경관생태적 개선방안)

  • Cho, Hyun-Ju;Ra, Jung-Hwa;Kim, Jin-Hyo
    • Current Research on Agriculture and Life Sciences
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    • v.29
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    • pp.11-19
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    • 2011
  • This research gives weight to establish practical improvement based on analysis of the landscape ecological character and value to realize the importance of small wetland and stream in cultivated areas functioning as a residual landscape element in rural landscape and to deal with ecological depression effectively. The results of summary are as follows. 1) The results of landscape ecological character analysis of wetlands biotop in total of 7 sites, for example, size of surface in site 5 was widely formed about $10,600m^2$, which was assessed satisfactory in terms of slope of waterfront, width of hedgerow, vegetation condition of composition and so on. Also, The number of flexibility showed 2 in site 8-1, the highest, and morphological diversity showed 1.47, the highest. 2) The results of problem analysis of wetland biotop, most of wetlands are analyzed that the width of hedgerow was below 1m. Also, the 4 wetlands in site 8 are appeared that hard to live slope vegetation in there because of slope of waterfronts are above $45^{\circ}$. 3) The results of landscape ecological character analysis of stream in total of 6 sites, for example, width of waterfront in site 4 showed 55m, the widest, and investigated consist of natural ingredients such as soil, rock, gravel. However, width of waterfront in site 2-2 showed 4m, the narrowest, and inclined angle of slope was formed a right angle. 4) The results of problem analysis of stream, width of waterfront hedgerow in site 2-1 showed about 5m, which was very narrower than width of waterfront, and toxic chemicals discharged from near cultivated area without any filtering. Also, all areas of site 2-2 was formed concrete, and was assessed dissatisfactory in terms of capacity of nature purification, flood control, habitat living space because of straight stream. 5) Based on the result above landscape ecological character and problem analysis, main improvement guidelines are set in terms of shape, vegetation, topography in case of wetlands, and which are set in terms of vertical, horizontal structure in case of stream.

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A study of the current(2003-2005) prevalence of anti-HBs and immunologic memory of hepatitis B vaccine in children from the central area of Korea (최근(2003-2005) 우리나라 중부지역 소아에서 B형 간염 항체 보유율과 백신의 면역학적 기억에 대한 연구)

  • An, Young Won;Chung, Eun Hee;Rheem, Insoo
    • Clinical and Experimental Pediatrics
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    • v.49 no.6
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    • pp.630-634
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    • 2006
  • Purpose : This study was conducted to assess the current(2003-2005) prevalence of anti-HBs and immunologic memory for Hepatitis B vaccine in children from the central area of Korea. Methods : Subjects were chosen from children and adolescents who received tests for hepatitis B surface antigen(HBsAg) and anti-HBs at Dankook University Hospital from March 2003 to May 2005. Among these, antibodies to hepatitis B core antigen(IgG anti-HBc) were checked. A single booster vaccination was performed on children whose anti-HBs titers were under 10 mIU/mL. One month after booster vaccination we rechecked the anti-HBs titer. Results : A total of 3,277 subjects were tested for HBsAg/anti-HBs, and 1,913(58.4 percent) of them were positive for anti-HBs. Of these, 29 subjects(0.9 percent) were positive for HBsAg. Positive results for anti-HBs by age were 78.6 percent for 6-12 months of age, 62.7 percent for 1-3 years of age, 51.9 percent for 4-6 years of age, 49.5 percent for 7-12 years of age, 63.4 percent for 13-15 years of age and 72.2 percent for 16-18 years of age. The 80 subjects who were tested negative for HBsAg/anti-HBs received a single booster vaccine, 71 subjects were tested positive for antibodies. IgG anti-HBc titer was checked for 169 of the subjects, 5 subjects were positive. Conclusion : In our study, a significant anamnestic response was observed in 88.8 percent of children. This is believed to be a result of the relatively long immunologic memory effect of the hepatitis B vaccination in children from the central area of Korea.

Sensitization of TNFα and Agonistic FAS/CD95 Antibody-Induced Apoptosis by INFγ on Neuroblastoma Cells (신경모세포종에서 IFNγ에 의한 TNFα와 길항적 FAS/CD95항체 유도성 세포고사의 감작화)

  • Bang, Ho Il;Kim, Jong Duck;Choi, Du Young
    • Clinical and Experimental Pediatrics
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    • v.46 no.7
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    • pp.702-709
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    • 2003
  • Purpose : $IFN{\gamma}$ sentitizes many tumor cells to $TNF{\alpha}$ and FASL-mediated apoptosis by enhancing the expression of TNF or FAS/CD95 receptor and modulating the activation of caspase and Bcl-2 family. It has been reported that $IFN{\gamma}$ and $TNF{\alpha}$ synergistically caused differentiation and growth inhibition of neuroblastoma cells. Even though some neuroblastoma cell express FASR/FASL on the cell surface, they could not induce apoptosis by ligation of the FAS/CD95 receptor. But the treatment of $IFN{\gamma}$ is reported to induce apoptosis in some neuroblastoma cell lines through the CD95/CD95L autocrine circuit. In this study, we examined whether $IFN{\gamma}$ could affect $TNF{\alpha}$ and agonistic FAS/CD95 antibody(CH-11)-induced apoptosis against neuroblastoma cell lines that had shown diverse drug sensitivity and resistance. Methods : CHLA-15, CHLA-90 and LA-N-2 neuroblastoma cells were cultured in IMDM and treated with recombinant $IFN{\gamma}$, $TNF{\alpha}$ and CH-11 antibody. Cell viability was measured by DIMSCAN with a fluorescent calcein-AM. Apoptosis was analyzed through flow cytometry using Annexin V-PE and 7-ADD staining and confirmed by pancaspase and caspase-8 blocking experiments. The expression of TNF RI and FAS/CD95 receptor was evaluated by flow cytometry using the corresponding antibody and PE-conjugated secondary antibody. Results : $IFN{\gamma}$ or $TNF{\alpha}$ alone had no demonstrable cytotoxic effects, whereas both cytokines in combination induced apoptosis synergistically in CHLA-15 and CHLA-90 cells. Although there was no cytotoxicity with the ligation of CH-11 alone in CHLA-90 cells, pretreatment of $IFN{\gamma}$ increased the sensitivity of CH-11-mediated apoptosis. The expression of TNFRI and FAS/CD95R were non-specifically enhanced after treatment of $IFN{\gamma}$ without relation to sensitivity to $TNF{\alpha}$ and CH-11. This finding suggest up-regulation of both receptors may contribute to sensitization of $TNF{\alpha}$ and CH-11-mediated apoptosis by $IFN{\gamma}$ in only sensitive cell lines. Conclusion : $IFN{\gamma}$ induced sensitization of $TNF{\alpha}$ and agonistic FAS/CD95 antibody-mediated apoptosis on some neuroblastoma cells through up-regulation of TNFRI and FAS/CD95 receptor.

Effect of Exercise Training on Aging Atrophyin Rat Skeletal Muscle III. Effect of Short Term Exercise Training for Senile Rat (흰쥐 골격근의 노화성 위축에 대한 운동훈련의 영향 III.노화 흰쥐에 적용한 단기간의 운동훈련의 영향)

  • 박승한;박원학;정형재
    • Biomedical Science Letters
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    • v.2 no.1
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    • pp.91-108
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    • 1996
  • The present study was designed to examine effect of short term treadmill and weight-training on aging arophy in the rat skeletal muscle. Male rats of 24 months old were used. Each groups included control, treadmill and weight-training for 4 weeks by using treadmill apparatus and body press apparatus. The histo and cytochemical, ultrastructural and stereological changes in senile skeletal muscles of the rat were observed in the present study. During the training period the body weight and muscular weight in all groups remained constant. The volume density of muscle fiber type IIC and IIB were increased, that of type IIA was decreased, but type I remained constant in treadmill-training group. In weight-training rat, the density of type IIA and IIB were increased, both those of type IIC was decreased. But, all changes of muscle fiber type is not significant. Senile control group some usual formation of mild contraction band, liposuscin pigment and muscular splitting were observed. After treadmill-training, histological and ultrastructural changes occurred in the muscle fiber, such as irregularity of the sarcolemma, interfibrillar vacuolization, longitudinal splitting, and widened I-bond. After weight-training, the changes occurred in the trained muscle fiber, such as appearances of many lysosomes and autophagic vacuoles, severe contraction band, and breakup of myofibrils. Histo and cytochemical studies showed that the activities of succinic dehydrogenase and acid phosphatase remained constant, activities of $Mg^{++}$-ATPase decrease with training. Stereological changes were not observed in the volume and numerical density of all subject component, but the surface density of mitochondrial inner membrane was increased with treadmill-training. These experimental results suggested that endurance training during short-term may result in the adaptible response in senile skeletal muscles. On the other side, weight-training is bad for senile skeletal muscle.

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Two Dimensional Size Effect on the Compressive Strength of Composite Plates Considering Influence of an Anti-buckling Device (좌굴방지장치 영향을 고려한 복합재 적층판의 압축강도에 대한 이차원 크기 효과)

  • ;;C. Soutis
    • Composites Research
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    • v.15 no.4
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    • pp.23-31
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    • 2002
  • The two dimensional size effect of specimen gauge section ($length{\;}{\times}{\;}width$) was investigated on the compressive behavior of a T300/924 $\textrm{[}45/-45/0/90\textrm{]}_{3s}$, carbon fiber-epoxy laminate. A modified ICSTM compression test fixture was used together with an anti-buckling device to test 3mm thick specimens with a $30mm{\;}{\times}{\;}30mm,{\;}50mm{\;}{\times}{\;}50mm,{\;}70mm{\;}{\times}{\;}70mm{\;}and{\;}90mm{\;}{\times}{\;}90mm$ gauge length by width section. In all cases failure was sudden and occurred mainly within the gauge length. Post failure examination suggests that $0^{\circ}$ fiber microbuckling is the critical damage mechanism that causes final failure. This is the matrix dominated failure mode and its triggering depends very much on initial fiber waviness. It is suggested that manufacturing process and quality may play a significant role in determining the compressive strength. When the anti-buckling device was used on specimens, it was showed that the compressive strength with the device was slightly greater than that without the device due to surface friction between the specimen and the device by pretoque in bolts of the device. In the analysis result on influence of the anti-buckling device using the finite element method, it was found that the compressive strength with the anti-buckling device by loaded bolts was about 7% higher than actual compressive strength. Additionally, compressive tests on specimen with an open hole were performed. The local stress concentration arising from the hole dominates the strength of the laminate rather than the stresses in the bulk of the material. It is observed that the remote failure stress decreases with increasing hole size and specimen width but is generally well above the value one might predict from the elastic stress concentration factor. This suggests that the material is not ideally brittle and some stress relief occurs around the hole. X-ray radiography reveals that damage in the form of fiber microbuckling and delamination initiates at the edge of the hole at approximately 80% of the failure load and extends stably under increasing load before becoming unstable at a critical length of 2-3mm (depends on specimen geometry). This damage growth and failure are analysed by a linear cohesive zone model. Using the independently measured laminate parameters of unnotched compressive strength and in-plane fracture toughness the model predicts successfully the notched strength as a function of hole size and width.

Skin Damage Sustained During Head-and-Neck and Shoulder Radiotherapy Due to the Curvature of Skin and the Use of Immobilization Mask (머리-목 그리고 어깨의 방사선 치료 시 피부곡면과 고정장치로 인한 피부손상연구)

  • Kim, Soo-Kil;Jeung, Tae-Sig;Lim, Sang-Wook;Park, Yeong-Mouk;Park, Dahl
    • Progress in Medical Physics
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    • v.21 no.1
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    • pp.86-92
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    • 2010
  • The purpose of this study was to measure curvature contour skin dose using radiochromic film and TLD for a conventional open field. We also attempted to quantify the degradation of skin sparing associated with use of immobilization devices for high energy photon beams and to calculate the skin dose with a help of Monte Carlo (MC) simulation. To simulate head-and-neck and shoulder treatment, a cylindrical solid water phantom 11 cm in diameter was irradiated with 6 MV x-rays using $40{\times}40\;cm^2$ field at 100 cm source axis distance (SAD) to the center of the phantom. Aquaplastic mesh mask was placed on the surface of the cylindrical phantom that mimicked relevant clinical situations. The skin dose profile was obtained by taking measurements from $0^{\circ}$ to $360^{\circ}$ around the circumference of the cylindrical phantom. The skin doses obtained from radiochromic film were found to be 47% of the maximum dose of $D_{max}$ at the $0^{\circ}$ beam entry position and 61% at the $90^{\circ}$ oblique beam position without the mask. Using the mask (1.5 mm), the skin dose received was 59% at $0^{\circ}$ incidence and 78% at $80^{\circ}$ incidence. Skin dose results were also gathered using thin thermoluminescent dosimeters (TLD). With the mask, the skin dose was 66% at $0^{\circ}$ incidence and 80% at $80^{\circ}$ incidence. This method with the mask revealed the similar pattern as film measurement. For the treatments of the head-and-neck and shoulder regions in which immobilization mask was used, skin doses at around tangential angle were nearly the same as the prescription dose. When a sloping skin contour is encountered, skin doses may be abated using thinner and more perforated immoblization devices which should still maintain immoblization.

The Levels of IgG Subclasses and Vaccine-induced Viral Antibodies in Childhood Minimal Change Nephrotic Syndrome (소아 신증후군에서 IgG 아군 및 바이러스 항체 값)

  • Song Yong-Hoon;Lee Kyung-Yil;Kim Dong-Un;Koh Dae-Kyun;Lee Byung-Churl
    • Childhood Kidney Diseases
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    • v.9 no.2
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    • pp.143-148
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    • 2005
  • Purpose : Hypogammaglobulinemia has been observed in nephrotic syndrome, but its pathophysiology remains unknown. We evaluated serum immunoglobulins, IgG subclasses, and vaccine-induced viral antibodies(anti-hepatitis B surface IgG and anti-measles IgG) in children with minimal change nephrotic syndrome(MCNS). Methods : Using the stored sera, the levels of immunoglobulin(IgC, IgM, IgA, and IgC) and IgG subclasses(IgG 1, 2, ,3, and 4), anti-HBs Ab and anti-measles IgG of 21 children with MCNS were analyzed and compared to those of 25 age-matched healthy children. Results : The mean values of IgG and IgG1 were $390{\pm}187\;mg/dL$ and $287{\pm}120\;mg/dL$ in nephrotic children, and $1,025{\pm}284\;mg/dL$ and $785{\pm}19\;mg/dL$ in control children, respectively. The values of the total IgG and the 4 IgG subclasses in nephrotic children were all significantly depressed(P<0.001), but the IgM($251{\pm}183\;mg/dL\;vs. 153{\pm}55\;mg/dL$, P=0.02) and IgE values(P=0.01) were elevated, and the IgA values were not changed. The seropositivity of anti-HBs IgG was 42.9$\%$(9 of 21 cases) in the MCNS group and 52$\%$(13/25) in the control group, and that of anti-measles IgG was 75$\%$(16/21) and 92$\%$(23/25), respectively, but there was no statistical difference between the two groups. Conclusion : IgG and IgG subclass levels in MCNS children are all depressed without significant seronegativity of the vaccine-induced viral antibodies. Further studies are needed to resolve the cause of hypogammaglobulinemia in MCNS. (J Korean Soc Pediatr Nephrol 2005;9:143-148)

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Morphological Characteristics and Germination as Affected by Low Temperature and GA in Orostachys 'Jirisan' and 'Jejuyeonhwa' Seeds, Korea Native Plant (지리산바위솔과 제주연화바위솔 종자의 형태특성 및 저온과 GA에 대한 발아 반응)

  • Kang, Jeong-Hee;Jeong, Kyeong-Jin;Choi, Kyoung-Ok;Chon, Young-Shin;Yun, Jae-Gill
    • Horticultural Science & Technology
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    • v.28 no.6
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    • pp.913-920
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    • 2010
  • This study was conducted to improve the seed germination by low temperature and $GA_3$ in $Orostachys$ $japonicus$ A. Berger (Jirisan) and $O.$ $iwarenge$ (Mak.) Hara (Jejuyeonhwa), Korean native plants. Observation of the seeds using a stereo microscope showed that all seeds of the two species have wrinkled surface and oblong shape. Seed size ranged 0.77-1.00/0.25-0.37 mm (length/width), indicating that the seeds are minute seeds. When the seeds of two $Orostachsis$ species were sown into petri-dish and placed in a plant growth chamber of 10, 15, 20, or $25^{\circ}C$, 'Jirisan' showed seed germination below 20% at all temperatures and 'Jejuyeonhwa' 80% at only $10^{\circ}C$. Seed germination of 'Jirisan' increased up to 44% at $10^{\circ}C$ by low temperature ($4^{\circ}C$) storage for 10 days, but decreased again at storage for more than 20 days. The seeds of 'Jejuyeonhwa' showed a large increase in seed germination by low temperature for 20-30 days, which was 95% at $10^{\circ}C$, but low temperature for more than 40 days significantly decreased seed germination. Dipping treatment in GA3 solution of $50-400mg{\cdot}L^{-1}$ for different periods (3, 6, 12, and 24 hrs) remarkably improved germination rate and speed in both species, 80-100% in 'Jirisan' and 90-100% in 'Jejuyeonhwa' at all concentrations and dipping times used in this study.

Determination of Chimera Types and Ploidy Level of Sports from 'Campbell Early' Grape (Vitis labruscana) (포도 '캠벨얼리' 품종에서 발생한 아조변이체의 배수성 및 키메라 형태 검정)

  • Noh, Jung-Ho;Park, Kyo-Sun;Yun, Hae-Keun;Do, Gyung-Ran;Hur, Youn-Young;Kim, Seung-Hui;Lee, Han-Chan;Ryou, Myung-Sang;Park, Seo-Jun;Jung, Sung-Min
    • Horticultural Science & Technology
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    • v.28 no.6
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    • pp.996-1002
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    • 2010
  • Flow cytometry (FCM) was used to measure the ploidy level of three different sports from 'Campbell Early' ($Vitis$ $labruscana$) grape. Results of the study showed different ploidy levels. FCM analysis for 'Campbell Early' grape which contains 2C DNA diploid cells showed single peak around 35-40 while 'Kyoho' grape with 4C DNA tetraploid cells had a different level of 70-80. However, analysis of the sports displayed a histogram with 2 peaks containing both 2C and 4C nuclei. There was no difference in histograms of 2C DNA flesh and pericarp; on the other hand, 4C DNA flesh type of sports had a different histogram from that of the 2C DNA pericarp. Chromosome numbers of diploid ('Campbell Early'), tetraploid ('Kyoho'), and three sports were counted under the microscope. 'Campbell Early' and 'Kyoho' have 38 and 76 chromosomes, respectively. Three different sports are mixoploids with mixtures of diploid and tetraploid cells. Microscopic observations of shoot apical meristems in sports from 'Campbell Early' grape were carried out to determine the type of plant chimera. 'Campbell Early' grape (diploid) and 'Kyoho' grape (tetraploid) showed that both had 2 tunica layers covering corpus cells, while the three different sports had tunica layers showing mostly oblique division. Most cells from 'Kyoho' grape were larger than 'Campbell Early' grape. Cells from L-2 and L-3 layers of the three sports were similar to 'Kyoho' grape in size, although all cells in L-1 surface layer were uniform in size like 'Campbell Early' grape. Results of FCM analysis indicated that both normal and polyploid cells could be intermixed in sports and could become mixoploidy consisting of diploid and tetraploid. All sports used in the tests were periclinal chimera plants with two distinct L-1 and L-2 cell layers. The result of this study suggests that all three sports which originated from 'Campbell Early' grape might be 2-4-4 type chimera formation.