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ABSTRACT: The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied
in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells
were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the
methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated
NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect
of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.
Science in China Series C Life Sciences 04/2012; 50(5):605-610. · 1.61 Impact Factor
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ABSTRACT: To verify the potential of the recombinant adeno-associated virus 2 (rAAV2) vector as a strategy for human transforming growth factor beta1 (hTGF-beta1) gene transfer in degenerative intervertebral discs of rabbit, to investigate the gene transduction efficacy and to quantify the biologic effects on the proteoglycan level after gene transferring.
Rabbit models of disc degeneration were established by injecting the 25 microL fibronectin fragment (Fn-f, 1 mmol/L), 4 weeks later, saline with or without virus was injected directly into 96 lumbar discs of 24 mature New Zealand white rabbits (male or female and weighing 1.7-2.2 kg) which were divided into 3 groups (n = 8). Group A received the 25 microL rAAV2-hTGF-beta1 (1 x 10(12) vg/mL); group B received rAAV2-enhanced green fluorescent protein (rAAV2-EGFP); and group C received PBS. Two rabbits of groups A, C were killed 1 week after injection, the immunohistochemical staining for hTGF-beta1 was performed on the slices of nucleus pulposus (NP) tissues. At 4, 8, and 12 weeks after gene transferring, NP tissues were harvested and cultured to quantify the changes of the proteoglycan level using 35S-sulfate incorporation assay. The expression of EGFP in group B was observed 12 weeks after injection.
Immunohistochemical staining showed that extensive and intense positive immunohistochemical staining for hTGF-beta1 were seen in group A when compared with group C 1 week after gene transferring. The nucleus pulposus tissues from the group A exhibited an increased synthesis of proteoglycan, which was significantly more than that from groups B and C (P < 0.05), and no significant difference was observed between group B and group C. The expression of EGFP in group B was high at 12 weeks.
The discs injected with rAAV2-hTGF-beta1 can highly expressed the therapeutic proteins for more than 12 weeks, it is suggested that rAAV2 should be an valid vector for transferring exogenous genes in the degenerative disc. The therapeutic factors hTGF-beta1 can efficiently increase the proteoglycan synthesis of the degenerative NP cells.
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 05/2010; 24(5):618-21.
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ABSTRACT: The soybean aphid (Aphis glycines Matsumura) is an important pest on soybean [Glycine max (L.) Merr.] in North America. Aphid resistance has recently been found on plant introduction (PI) 567543C, but little is known about its genetic control. The objectives of this study were to identify the resistance genes in PI 567543C with molecular markers and validate them in a different genetic background. A mapping population of 249 F(4) derived lines from a cross between PI 567543C and a susceptible parent was investigated for aphid resistance in both the greenhouse and the field. The broad sense heritability of aphid resistance in the field trial was over 0.95. The segregation of aphid resistance in this population suggests a major gene controlling the resistance. Bulked segregant analysis with molecular markers revealed a potential genomic region. After saturating this putative region with more markers, a genetic locus was mapped in an interval between Sat_339 and Satt414 on chromosome 16 (linkage group J) using the composite interval mapping method. This locus explained the majority of the phenotypic variation ranging from 84.7% in the field trial to 90.4% in the greenhouse trial. Therefore, the aphid resistance in PI 567543C could be mainly controlled by this gene. This aphid resistance gene was mapped on a different chromosome than the other resistance genes reported previously from other resistant germplasms. This gene appears to be additive based on the aphid resistance of the heterozygous lines at this locus. Thus, a new symbol Rag3 is used to designate this gene. Moreover, Rag3 was confirmed in a validation population. This new aphid-resistance gene could be valuable in breeding aphid resistant cultivars.
Theoretical and Applied Genetics 04/2010; 120(6):1183-91. · 3.30 Impact Factor
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ABSTRACT: Seed calcium content is an important quality attribute of specialty soybean [Glycine max (L.) Merr.] for soyfoods. However, analyzing seed for calcium content is time consuming and labor intensive. Knowing quantitative trait loci (QTL) for seed calcium will facilitate the development of elite cultivars with proper calcium content through marker-assisted selection (MAS). The objective of this study was to identify major QTL associated with calcium content in soybean seed. Calcium content was tested in 178 F(2:3) and 157 F(2:4) lines derived from the cross of SS-516 (low calcium) x Camp (high calcium). The F(2:3) lines were genotyped with 148 simple sequence repeat markers in a previous study on seed hardness, and the genotypic data were used in the QTL analysis of the current study. Four QTL designated as Ca1, Ca2, Ca3, and Ca4 on linkage groups (LGs) A2, I, and M were identified by both single-marker analysis and composite-interval mapping, and the QTL accounted for 10.7%, 16.3%, 14.9%, and 9.7% of calcium content variation, respectively. In addition, multiple-interval mapping analysis revealed a significant dominant-by-dominant interaction effect between Ca1 and Ca3, which accounted for 4.3% calcium content variation. These QTL will facilitate the implementation of MAS for calcium content in soybean-breeding programs.
The Journal of heredity 12/2008; 100(2):263-9. · 2.05 Impact Factor
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ABSTRACT: The soybean aphid (Aphis glycines Matsumura) is an important pest of soybean [Glycine max (L.) Merr.] in North America since it was first reported in 2000. PI 567541B is a newly discovered aphid resistance germplasm with early maturity characteristics. The objectives of this study were to map and validate the aphid resistance genes in PI 567541B using molecular markers. A mapping population of 228 F3 derived lines was investigated for the aphid resistance in both field and greenhouse trials. Two quantitative trait loci (QTLs) controlling the aphid resistance were found using the composite interval mapping method. These two QTLs were localized on linkage groups (LGs) F and M. PI 567541B conferred resistant alleles at both loci. An additive x additive interaction between these two QTLs was identified using the multiple interval mapping method. These two QTLs combined with their interaction explained most of the phenotypic variation in both field and greenhouse trials. In general, the QTL on LG F had less effect than the one on LG M, especially in the greenhouse trial. These two QTLs were further validated using an independent population. The effects of these two QTLs were also confirmed using 50 advanced breeding lines, which were all derived from PI 567541B and had various genetic backgrounds. Hence, these two QTLs identified and validated in this study could be useful in improving soybean aphid resistance by marker-assisted selection.
Theoretical and Applied Genetics 12/2008; 118(3):473-82. · 3.30 Impact Factor
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ABSTRACT: Linkage maps of the sweet cherry cultivar ‘Emperor Francis’ (EF) and the wild forest cherry ‘New York 54’ (NY) were constructed
using primarily simple sequence repeat (SSR) markers and gene-derived markers with known positions on the Prunus reference map. The success rate for identifying SSR markers that could be placed on either the EF or NY maps was only 26%
due to two factors: a reduced transferability of other Prunus-species-derived markers and a low level of polymorphism in the mapping parents. To increase marker density, we developed
four cleaved amplified polymorphic sequence markers (CAPS), 19 derived CAPS markers, and four insertion–deletion markers for
cherry based on 101 Prunus expressed sequence tags. In addition, four gene-derived markers representing orthologs of a tomato vacuolar invertase and
fruit size gene and two sour cherry sorbitol transporters were developed. To complete the linkage analysis, 61 amplified fragment
length polymorphism and seven sequence-related amplified polymorphism markers were also used for map construction. This analysis
resulted in the expected eight linkage groups for both parents. The EF and NY maps were 711.1cM and 565.8cM, respectively,
with the average distance between markers of 4.94cM and 6.22cM. A total of 82 shared markers between the EF and NY maps
and the Prunus reference map showed that the majority of the marker orders were the same with the Prunus reference map suggesting that the cherry genome is colinear with that of the other diploid Prunus species.
Tree Genetics & Genomes 09/2008; 4(4):897-910. · 2.34 Impact Factor
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ABSTRACT: The soybean aphid (Aphis glycines Matsumura) has become a major pest of soybean in North America since 2000. Seven aphid resistance sources, PI 71506, Dowling, Jackson, PI 567541B, PI 567598B, PI 567543C, and PI 567597C, have been identified. Knowledge of genetic relationships among these sources and their ancestral parents will help breeders develop new cultivars with different resistance genes. The objective of this research was to examine the genetic relationships among these resistance sources. Sixty-one lines were tested with 86 simple sequence repeat (SSR) markers from 20 linkage groups. Non-hierarchical (VARCLUS) and hierarchical (Ward's) clustering and multidimensional scaling (MDS) were used to determine relationships among the 61 lines. Two hundred and sixty-two alleles of the 86 SSR loci were detected with a mean polymorphism information content of 0.36. The 61 lines were grouped into 4 clusters by both clustering methods and the MDS results consistently corresponded to the assigned clusters. The 7 resistance sources were clustered into 3 different groups corresponding to their geographical origins and known pedigree information, indicating genetic differences among these sources. The largest variation was found among individuals within different clusters by analysis of molecular variance.
Genome 01/2008; 50(12):1104-11. · 1.65 Impact Factor
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ABSTRACT: T he soybean aphid (SBA; Aphis glycines Matsumura) was fi rst discovered in eight midwestern U.S. states in 2000. Since then it has spread throughout the north-central United States and parts of Canada (North Central Soybean Research Program, 2004) and has become one of the major pests aff ecting soybean production in North America. Soybean aphid populations can double very quickly (McCornack et al., 2004), reaching thou-sands of aphids per plant. Aphid feeding reduces photosynthesis (Macedo et al., 2003) and reduces yield components including plant height, number of nodes and pods per plant, seed size, and bean quality (DiFonzo and Hines, 2002; Ostlie, 2001). In effi cacy trials conducted in Michigan during SBA outbreak years, yield in untreated plots was 18 to 40% less than yield in treated plots (DiFonzo, 2006; DiFonzo and Hines, 2002). Insecticides are still the primary means of controlling SBA, increasing production costs and human exposure. In 2005, an outbreak year for SBA across the Midwest, millions of acres were treated (USDA-National Agricultural Statistics Service, 2006). Insecticide applications also kill natural enemies of SBAs (Smith and Krischik, 1999) and may fl are populations of other soybean pests such as spider mites. Host-plant resistance is the most eff ective means of control of insects. Soybeans resistant to SBA colonization ABSTRACT In a previous study, two soybean [Glycine max (L.) Merr.] plant introductions (PIs), PI 567541B and PI 567598B, were found to possess anti-biosis-type resistance to the soybean aphid (Aphis glycines Matsumura). Plants with anti-biosis resistance negatively interfere with the reproduction of the aphid and thus control the insect effectively. Field studies were conducted to determine the inheritance of antibiosis resis-tance in PI 567541B and PI 567598B. The two resistant PIs were crossed with one or two sus-ceptible soybean lines and the F 1 and F 2 plants and F 2:3 families were evaluated for aphid resis-tance. All F 1 plants were found to be suscep-tible to soybean aphids. The plants in seven F 2 populations segregated in a 15:1 susceptible/ resistant ratio, which is the expected ratio for a trait controlled by two recessive genes. The F 2:3 families also segregated in a 15:1 suscep-tible/resistant ratio. Therefore, the segregation data suggest that two major recessive genes are involved in the resistance in PI 567541B and PI 567598B. This information will be use-ful to breeders for designing effi cient breeding schemes for developing soybean cultivars with antibiosis resistance to aphids.
CROP SCIENCE. 01/2008; 48.
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ABSTRACT: The effects of AAV-TGFbeta(1) and AAV-TGFbeta(3) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFbeta(1) or AAV-TGFbeta(3), their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of (35)S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFbeta(1) and AAV-TGFbeta(3) could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFbeta(1) was better than that of AAV-TGFbeta(3). For the later dedifferentiated NP cells, the AAV-TGFbeta(3) could promote their synthesis, but AAV-TGFbeta(1) could slightly inhibit their synthesis. Therefore, AAV-TGFbeta(1) and AAV-TGFbeta(3) could be used for the earlier dedifferentiated NP cells, and the TGFbeta(3) could be used as the objective gene for the later dedifferentiated NP cells.
Science in China Series C Life Sciences 11/2007; 50(5):605-10. · 1.61 Impact Factor
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ABSTRACT: Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe; HG) is one of the most destructive pests of soybean (Glycine max (L.) Merr.) in the United States. Over 100 SCN-resistant accessions within the USDA Soybean Germplasm Collection have been identified, but little is known about the genetic diversity of this SCN-resistant germplasm. The objective of this research was to evaluate the genetic variation and determine the genetic relationships among SCN-resistant accessions. One hundred twenty-two genotypes were evaluated by 85 simple sequence repeat (SSR) markers from 20 linkage groups. Non-hierarchical (VARCLUS) and hierarchical (Ward's) clustering were combined with multidimensional scaling (MDS) to determine relationships among tested lines. The 85 SSR markers produced 566 allelic fragments with a mean polymorphic information content (PIC) value of 0.35. The 122 lines were grouped into 7 clusters by 2 different clustering methods and the MDS results consistently corresponded to the assigned clusters. Assigned clusters were dominated by genotypes that possess one or more unique SCN resistance genes and were associated with geographical origins. The results of analysis of molecular variance (AMOVA) showed that the variation differences among clusters and individual lines were significant, but the differences among individuals within clusters were not significant.
Genome 09/2006; 49(8):938-49. · 1.65 Impact Factor
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ABSTRACT: Gulf war illnesses (GWI) are currently affecting thousands of veterans. To date, the molecular mechanisms underlying the pathogenesis of these illnesses remain unknown. During Gulf war I, military personnel were exposed to multiple stressors, one or more vaccines, pyridostigmine (PY), and other chemicals. In our previous studies, we found that stress induces activation of mitogen activated protein-kinase kinase 4 (MKK4) and c-Jun-N-terminal kinase (JNK) in the mouse brain (Liu et al. 2004). Our working hypothesis is that stress, vaccination, and PY may synergistically induce activation of MKK4 and JNK in the brain, leading to over-activation of these kinases and neurological injuries. To test our hypothesis, we examined the effect of keyhole limpet hemocyanin (KLH) immunization alone or in combination with PY on activation of MKK4 and JNK induced by stress. We found that KLH immunization alone had a small effect on MKK4 or JNK activity but it significantly enhanced and prolonged activation of these kinases induced by stress, from a few hours to several days. Additionally, KLH immunization caused activation of p38MAPK. PY treatment further enhanced and prolonged activation of these kinases induced by stress in combination with KLH immunization and triggered activation of caspase-3. Our current studies suggest that stress, vaccination, and PY may synergistically act on multiple stress-activated kinases in the brain to cause neurological impairments in GWI.
Journal of Neurochemistry 06/2005; 93(4):1010-20. · 4.06 Impact Factor
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ABSTRACT: Stress is a part of daily life. However, molecular mechanisms underlying the activation of limbic-hypothalamic-pituitary-adrenal (LHPA) axis remains unknown. In this study, we explored whether activation of the mitogen-activated kinase kinase 4 (MKK4)-c-Jun-N-terminal kinase (JNK) signaling pathway may play a role in the activation of the LHPA axis. We found that forced-swim stress induced elevation of activated MKK4 in the hippocampal formation, amygdala, and hypothalamus. Unlike MKK4, a high basal level of JNK activity is present in many brain areas of unstressed mice. Forced-swim stress significantly elevated JNK activity in the hypothalamus and amygdala and, to a lesser extent, in the cortex, CA1 and CA3 regions, and the dentate gyrus. To further investigate the role of MKK4 and JNK in induction of stress responses, we investigated whether a different stress, namely, restraint stress, induced activation of MKK4 or JNK in the brain. We found that restraint stress also induced elevation of activated MKK4 and JNK in the hippocampal formation, amygdala, and hypothalamus. Because MKK4 and JNK were activated within 5 min following stress, we propose that the MKK4-JNK signaling may be an early neural event in the initiation of neuroendocrine, autonomic and behavioral stress responses.
Journal of Neurochemistry 06/2004; 89(4):1034-43. · 4.06 Impact Factor
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ABSTRACT: Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial beta-amyloid precursor protein APP-V642I (IAPP) or mutant presenilin-1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild-type amyloid precursor protein or presenilin-1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with beta-amyloid (Abeta) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a gamma-secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Abeta peptide induces actin stress fibers. Because activation of the p38 mitogen-activated protein kinase (p38MAPK)-mitogen-associated protein kinase-associated protein kinase (MAPKAPK)-2-heat-shock protein 27 signaling pathway mediates actin polymerization, we explored whether Abeta peptide activates p38MAPK and MAPKAPK-2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK-2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Abeta peptide, IAPP or mPS1. Moreover, treatment with a gamma-secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK-2. In summary, our data suggest that overproduction of Abeta peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.
Journal of Neurochemistry 12/2002; 83(4):828-36. · 4.06 Impact Factor
