Gerald Hankins - Academia.edu (original) (raw)
Papers by Gerald Hankins
International Journal of Molecular Sciences, Oct 25, 2023
Evolutionary Applications, 2009
Advances in neurobiology, 2024
Gene Therapy, Jul 7, 2005
This study was designed to see if immunosuppression achieved using local application of cyclospor... more This study was designed to see if immunosuppression achieved using local application of cyclosporine A (Cs. A) or CD4 and CD8 antibodies would improve bone formation following intramuscular injections of human BMP-4 and BMP-9 adenoviral vectors (ADhBMP4 and ADhBMP9) in Sprague-Dawley rats. Cs. A was injected into the thigh muscle. After 2 days, ADhBMP4, ADhBMP9, and the antibodies were separately injected into the left and right rear legs. At this time, the number of CD4+/CD3+ cells was significantly lower and the number of CD8+/CD3+ cells higher in the Cs. A group than in the control group (Po0.01). The total number of white blood cells 3 days following injection of CD4 and CD8 antibodies was significantly lower than that before the injection (Po0.01). At 4 weeks after the viral and antibody injections, mean bone volumes at the ADhBMP9 treatment sites were 0.2970.01 cm 3 in the viral control group, 0.1770.03 cm 3 in the Cs. A-ADhBMPs group, and 0.5970.07 cm 3 in the antibodies-ADhBMPs group. ADhBMP4 did not induce new bone formation in any group. This study demonstrates that local immunomodulation may improve the osteogenic potential of bone morphogenetic protein gene therapy in the clinical setting.
PubMed, May 4, 1995
The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIr) is required for the a... more The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIr) is required for the activation of transforming growth factor beta, and previously we have found its expression to be significantly reduced in both rat and human hepatocellular carcinomas (HCCs). Therefore, we have postulated that loss of the M6P/IGFIIr gene may be mechanistically involved in liver carcinogenesis. Using the polymerase chain reaction, we utilized two polymorphisms in the 3' untranslated region of the M6P/IGFIIr gene to screen non-cirrhotic, hepatitis virus negative patients with hepatocellular tumors for LOH. Twenty-two of 36 (61%) patients were informative (heterozygous), and 14/22 (64%) liver tumors had LOH; 11/16 (69%) carcinomas, 1/3 (33%) fibrolamellar tumors and 2/3 (67%) adenomas. This is the first report of LOH at the M6P/IGFIIr locus in human hepatocellular tumors, and the presence of LOH in adenomas suggests that allelic loss may be an early event in the etiology of HCCs. These results support the hypothesis that the M6P/IGFIIr gene may function as a tumor suppressor gene in the liver.
Interventional Neuroradiology, Mar 1, 1998
We determined the propensity for and reversibility of transforming growth factor-f3 (TGFf3) bindi... more We determined the propensity for and reversibility of transforming growth factor-f3 (TGFf3) binding to uncoated Guglielmi Detachable Coils (GDC) and to GDC coated with extracellular matrix (ECM) proteins. Three 1.0 centimetre samples each of uncoated GDC-18 and of GDC-18 coated with either poly-L-lysine, laminin, type I collagen, type IV collagen, fibronectin, or poly-L-lysine and laminin were prepared. These samples were immersed briefly in a solution containing [125_ labelled TGFf3 at a concentration of 0.225 pg/ml with initial specific activity of 123.3 mCilmg (DuPont-NEN, Billerica, MA), and were counted using a scintillation counter. Each sample was then placed in a vial containing saline, shaken for 60 seconds, and counted again. Selected samples were immersed for varying periods within the TGFf3 solution and counted before and after saline rinse. Samples were rinsed one week after initial rinsing and counted again. The amount of binding between coil types was compared using the Student t test. For all samples initial binding ofTGFf3 was in the order of 60-120 pg!cm. For the pre-rinse data there were no statistically significant differences between the amount bound to any single coil coating type relative to other coatings. Compared to the initial accumulations, the amount remaining after rinsing ranged from 40% (poly-L-lysine) to 63% (poly-L-lysine with laminin), with a mean of 55% among the seven coil types. After rinsing there was more growth factor remaining on uncoated coils than on poly-L-lysine-coated coils (p=0.05), fibronectin-coated coils (p=O.Ol), and type IV collagen-coated coils (p=O.04). There was a trend toward greater residual growth factor on coils coated with poly-L-lysine and laminin compared to coils coated with poly-L-lysine alone (p=O.lO). Delayed, second rinsing of the samples one week after initial testing demonstrated only minor incremental loss of TGFf3 from the coil surfaces. After five minutes of immersion, accumulation was approximately 200% greater than that noted with brief submersion, but immersions lasting over five minutes did not yield increasing levels ofTGFf3 binding. TGFf3 binds to GDC coils. Binding is not improved with ECM protein-coated coils compared to uncoated coils. The absolute amount of TGFf3 bound to the coil will likely result in local concentrations of growth factor in the order of those required for biological activity in vivo.
Genetics, Oct 1, 1995
We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis... more We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.
Neurosurgery, Apr 1, 2003
There are anatomical and functional differences between human dental pulp (DP) and periodontal li... more There are anatomical and functional differences between human dental pulp (DP) and periodontal ligament (PDL). However, the molecular biological differences and function of these tissues are poorly understood. In the present study, we employed a cDNA microarray array to screen for differentially expressed genes (DEGs) between human DP and PDL tissues, and used the online software WebGestalt to perform the functional analysis of the DEGs. In addition, the STRING database and KEGG pathway analysis were applied for interaction network and pathway analysis of the DEGs. DP and PDL samples were obtained from permanent premolars (n=16) extracted for orthodontic purposes. The results of the microarray assay were confirmed by RT-qPCR. The DEGs were found to be significantly associated with the extracellular matrix and focal adhesion. A total of 10 genes were selected to confirm the results. The mRNA levels of integrin alpha 4 (ITGA4), integrin alpha 8 (ITGA8), neurexin 1 (NRXN1) and contactin 1 (CNTN1) were significantly higher in the DP than in the PDL tissues. However, the levels of collagen type XI alpha 1 (COL11A1), aggrecan (ACAN), collagen type VI alpha 1 (COL6A1), chondroadherin (CHAD), laminin gamma 2 (LAMC2) and laminin alpha 3 (LAMA3) were higher in the PDL than in the DP samples. The gene expression profiles provide novel insight into the characterization of DP and PDL tissues, and contribute to our understanding of the potential molecular mechanisms of dental tissue mineralization and regeneration.
The Journal of Immunology
Oil from Nigella sativa seeds (“black seed oil”) has been used for health care and medical purpos... more Oil from Nigella sativa seeds (“black seed oil”) has been used for health care and medical purposes for over 2000 years. The major active compound thymoquinone (TQ) has been shown to have immune regulatory effects in various diseases. Tumor microenvironment is critical for tumor development. Immune cells, particularly T cells, are central players to kill tumor cells and affect tumor progression and metastasis. The impact of TQ on human T cell-mediated anti-tumor response was investigated. Using human PBMCs, we activated T cells with anti-CD3/CD28 in vitro, and treated cells with TQ. First, we tested the impact of TQ on T cell activation, proliferation and apoptosis; second, the anti-tumor effect of T cells after treatment with TQ; last, we determined alterations in T cells gene expression at the RNA level after TQ treatment. TQ treatment increased activation and proliferation of T cells at days 2–4 and more activation-induced cell death at day 3. After 7–11 days, TQ-treated cells sh...
Radiology, 1998
To demonstrate in vivo that platinum embolic coils can be used to deliver genetically modified, g... more To demonstrate in vivo that platinum embolic coils can be used to deliver genetically modified, growth factor-secreting fibroblast grafts into the endovascular space with the long-term goal of improving fibrosis within coil-embolized cerebral aneurysms. Murine fibroblasts that contained multiple inserts of the DNA for human basic fibroblast growth factor were grown in culture onto 10-mm-long segments of Guglielmi detachable coils. Control (n = 4) and fibroblast-bearing (n = 4) coils were implanted into the common carotid artery in nude rats. The arterial segments that contained the coil were harvested after 14 or 35 days. Cellular content and collagen formation in the treated vessels were assessed histologically. At both 14 and 35 days, samples with control coils showed primarily involuting blood elements with minimal fibroblast proliferation or collagen formation. At 14 days, samples with fibroblast-bearing coils showed extensive fibroblast proliferation. At 35 days, samples with fibroblast-bearing coils showed marked interval fibroblast proliferation and collagen formation. Platinum coils can be used as a cell delivery device. Direct intravascular implantation of growth factor-secreting fibroblast grafts leads to improved intravascular scar formation, therefore theoretically reducing the potential for aneurysm regrowth or coil compaction.
Radiology, 1998
To evaluate the growth and adhesion characteristics in vitro of genetically modified, basic fibro... more To evaluate the growth and adhesion characteristics in vitro of genetically modified, basic fibroblast growth factor-producing fibroblasts on platinum detachable coils. Coils of two sizes were coated with laminin, poly-L-lysine, fibronectin, and type I and type IV collagen and were cultured with basic fibroblast growth factor-secreting fibroblasts. Type I collagen strands were inserted in the lumen of some coils. Cellular proliferation and adherence during passage of coils through microcatheters were studied with both light and scanning electron microscopy. Growth factor concentration in the culture medium was measured. Rapid cellular proliferation was noted on all coated coils except those coated with type IV collagen. Proliferation on uncoated coils was slightly slower than on most coated coils, although confluent cell layers were present on uncoated larger-diameter coils within 48 hours. Cells had a marked propensity to grow between the primary coil windings into the coil lumen, except in coils that contained collagen filaments. Passage through microcatheters caused widespread stripping of cells from the outer surface of coils, especially the uncoated samples. Viable cells remained in the coil lumen. Supernatant contained high concentrations of growth factor. Platinum embolic coils are a promising mechanism of cell delivery for stimulation of scar formation or other desirable biologic effects.
Proceedings of the West Virginia Academy of Science, Jul 26, 2016
Response of glioblastoma cells to activation of the G-protein coupled estrogen receptor, GPER1/GP... more Response of glioblastoma cells to activation of the G-protein coupled estrogen receptor, GPER1/GPR30, and possible crosstalk with the aryl hydrocarbon receptor. Glioblastomas are almost invariably fatal brain tumors. Glioblastoma incidence in men is 1.5 times that of pre-menopausal women and this difference decreases postmenopause. Therefore estrogen is thought to play a protective role, although the mechanism is not well understood. Glioblastomas produce and respond to a tryptophan derivative, kynurenine, that binds and activates the aryl hydrocarbon receptor (AHR). Promiscuity of ligand binding can result in cross talk between AHR and nuclear estrogen receptors and both receptor types are known to use the p300 acetyltransferase as a cofactor. We demonstrated that inhibition of p300 resulted in a reduction of proliferation of CH157-MN meningioma cells. Treatment with βestradiol or kynurenine partially abrogated this effect, suggesting both ligands may act through another receptor that we hypothesize to be the G-protein linked estrogen receptor, GPER1/GPR30. We verified the expression of GPER1, estrogen receptor β; some estrogen receptor β isoforms and AHR by CH157-MN cells and U-87 and A-172 glioblastoma cells. Inhibition of p300 with C646 reduced proliferation of both U87 and A172 cells. In U87 cells, this effect was abrogated by co-treatment with βestradiol, the xenobiotic AHR agonist 2',3',7,8'-tetrachlorodibenzo-p-dioxin, and the GPER1 agonist G-1. Exogenous kynurenine did not abrogate the effect of p300 inhibition. However, in A172 cells, none of the agonists abrogated the effect of p300 inhibition, possibly due to the higher kynurenine production in A172 cells. Treatment of A172 and U87 cells with G-1 resulted in decreased cell proliferation which was abrogated by co-treatment with the GPER1 antagonist G-36.
<p>Different controls with different levels of ethanol, the solvent used for dissolving cap... more <p>Different controls with different levels of ethanol, the solvent used for dissolving capsaicinoids in the study, were tested at 24 and 72 H. Control-1 contains culture media without ethanol. All other controls contain culture media with different levels of ethanol. Control-2, -3, -4 and -5 contained 0.2, 0.4, 0.6 and 0.8% ethanol, respectively. These were the corresponding controls for 100, 200, 300 and 400 μM capsaicin as well as dihydrocapsaicin treatments; and 0.75, 1.50, 2.25 and 3.00 g L<sup>-1</sup> ghost pepper treatments, respectively. Control-6, -7, -8 and -9 contained 0.24, 0.48, 0.72 and 0.96% ethanol, respectively. These were the corresponding controls for 100 + 20; 200 + 40; 300 + 60; and 400 + 80 μM capsaicin + dihydrocapsaicin treatments, respectively (refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206183#pone.0206183.g002" target="_blank">Fig 2</a> for treatments). Values are mean ± SD; n = 4; NS = Not significantly different from control-1 (<i>p</i> ≤ 0.05).</p
Molecular Therapy, 2004
Although adenoviral vectors have a very broad range and high efficiency of cell transduction both... more Although adenoviral vectors have a very broad range and high efficiency of cell transduction both in vitro and in vivo, the uses of adenoviral vectors have been significantly limited in immunocompetent animals. We tested whether the amount of protein expression was the major factor determining the foreign gene functions. The recombinant adenoviral vectors of human bone morphogentic protein 4 and luciferase were used to answer this question. Direct viral injection and ex vivo methods were chosen in immunodefective athymic nude (AN) and immunocompetent SD rats. On Day 0, the primary rat bone marrow cells were infected with either ADhBMP4 or ADLUC at 107 PFU/flask or PBS. At the same time, five AN and SD rats were given a direct injection of either ADhBMP4 or ADLUC (107 PFU/100 μl) into the right thigh. On Day 1, both control and virally infected cells were injected into the left thigh with 2 × 106 cells/100 μl/flask. The rats underwent imaging performed with a cooled CCD before and after an intraperitoneal luciferin injection on Days 1 and 7. On Day 1, the densities of luminescent signals (×107 photons/sec) were the following: 1.83 ± 0.56 for SD rats with direct ADLUC injection; 3.73 ± 1.15 for SD rats with ex vivo ADLUC injection; 5.01 ± 1.91 for AN rats with direct ADLUC injection; and 11.9 ± 3.35 for AN rats with ex vivo ADLUC injection. The luminescent signals in the control groups were the same as background. By Day 7, the percentages of luminescence densities in ADLUC groups (compared with the same site on Day 1) were the following: 14.1% for SD rats with direct ADLUC injection; 1.06% for SD rats with ex vivo ADLUC injection; 25.4% for AN rats with direct ADLUC injection; and 0.8% for nude rats with ex vivo ADLUC injection. The animals were scanned with CT 1 month after injection. The bone volumes (cm3) of ADhBMP4 induced were 0.85 ± 0.08 for direct injection and 0.15 ± 0.03 for ex vivo in AN rats, and no bone for direct injection and 0.12 ± 0.02 for ex vivo in SD rats. No bone was observed at sites receiving injections of control cells or ADLUC. These data demonstrate that AN and SD rats display similar expression profiles of foreign gene expression delivered with adenoviral vectors, regardless of whether direct viral injection or the ex vivo method is used. As we know the amount of new bone is dose dependent in AN rats, and the average amount of bone induced by hBMP4 is smaller when the ex vivo method is employed than when direct viral injection is used. This result reflects the lower level of hBMP4 expression using the ex vivo method than directly viral injection. However, in SD rats, although hBMP4 expression may be lower after the ex vivo injection than after directly viral injection, the ex vivo ADhBMP4 delivery induces bone formation. These results indicate that the amount of BMP expression is not the major factor affecting the amount of new bone in SD rats. The major factors affecting the osteogenic potentials of ADhBMPs in SD rats may be some immune factors, which may have higher titer in direct adenoviral injection comparing to the ex vivo delivery.
Human Gene Therapy, 1999
Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standar... more Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standard rat in vivo assay systems. Previous studies have demonstrated the clinical utility of these proteins in spinal fusion, fracture healing, and prosthetic joint stabilization. Gene therapy is also a theoretically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expression may be possible in future. This study was performed to determine whether an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U87 MG cells transduced per cell with 20 MOI of an adenoviral construct containing the BMP-2 gene under the control of the universal CMV promoter (Ad-BMP-2) showed positive antibody staining for the BMP-2 protein at posttransfection day 2. The synthesis and secretion of active BMP-2 into the conditioned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 stromal cell assay. In vivo, Sprague-Dawley rats and athym ic nude rats were injected with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6, 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induced mesenchymal stem cell chemotaxis and proliferation, with subsequent differentiation to chondrocytes. The chondrocytes secreted a cartilaginous matrix, which then mineralized and was replaced by mature bone. This study demonstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone formation. However, in immunocompetent animals the endochond ral response is attenuated, secondary to the massive immune response elicited by the firstgeneration adenoviral construct.
Acta Neuropathologica, 2002
Tissue Engineering, 2006
The osteogenic potential of AAV5hBMP6 was compared with that of ADhBMP6 in immunodeficient and im... more The osteogenic potential of AAV5hBMP6 was compared with that of ADhBMP6 in immunodeficient and immunocompetent rats. AAV5hBMP6 (2.3 x 10(12) particles) and ADhBMP6 (5 x 10(7) PFU) elicited viral antibody production in immunocompetent rats. Among rats that received AAV5hBMP6, the earliest time points at which the bone was visible under CT scanner were 30 days in 2-month-old Sprague-Dawley (SD) rats and 60 days in 18-month-old SD rats. The mean volumes of ectopic bone 90 days after viral injection were 0.31 +/- 0.14 cm(3) in athymic nude rats, 0.64 +/- 0.12 cm(3) in 2-month-old SD rats, and 0.21 +/- 0.10 cm(3) in 18-month-old SD rats. In contrast, among rats that received ADhBMP6, the earliest time points to observe the bone formation by CT scan were 15 days in 2-month-old rats and no bone formation in 18-month-old SD rats. The mean volumes of ectopic bone were 4.17 +/- 0.05 cm(3) in athymic nude rats and 0.06 +/- 0.03 cm(3) in 2-month-old SD rats. Although both types of viruses induced an immune response in immunocompetent animals, this response played different roles in the process of bone formation induced by the BMP6 vectors.
Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Produ... more Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Production of capsaicin is an important quantitative trait with high environmental variance, so the development of markers regulating capsaicinoid accumulation is important for pepper breeding programs. In this study, we performed association mapping at the gene level to identify single nucleotide polymorphisms (SNPs) associated with capsaicin pathway metabolites in a diverse Capsicum annuum collection during two seasons. The genes Pun1, CCR, KAS and HCT were sequenced and matched with the whole-genome sequence draft of pepper to identify SNP locations and for further characterization. The identified SNPs for each gene underwent candidate gene association mapping. Association mapping results revealed Pun1 as a key regulator of major metabolites in the capsaicin pathway mainly affecting capsaicinoids and precursors for acyl moieties of capsaicinoids. Six different SNPs in the promoter sequence...
Glioma incidence in males is 1.5 times that of females, thus it is suspected that female sex horm... more Glioma incidence in males is 1.5 times that of females, thus it is suspected that female sex hormones play a protective role against gliomas. Compounds found in water contaminated by extractive industry waste can disrupt hormone mediated signaling. Studies have been done on endocrine disrupting effects on estrogen signaling, but little has been done on their effects on progesterone signaling. Additionally, the focus has been on classical receptors, with almost no investigation of G-protein coupled receptors. Here we investigate the effects of exposure to coal dust on cell proliferation and expression of progestin and adipoQ receptors (PAQRs) in two human glioma cell lines, A172 and U87. Treatments with coal dust and treatments with progesterone were performed. End point RT-PCR demonstrated that A172 cells express PAQR3, PAQR5, and PAQR9. This was the first demonstration of PAQR5 expression in A172 cells. U87 cells express PAQR3, PAQR5, PAQR7, PAQR8, and PAQR9. Real time RT-PCR was p...
International Journal of Molecular Sciences, Oct 25, 2023
Evolutionary Applications, 2009
Advances in neurobiology, 2024
Gene Therapy, Jul 7, 2005
This study was designed to see if immunosuppression achieved using local application of cyclospor... more This study was designed to see if immunosuppression achieved using local application of cyclosporine A (Cs. A) or CD4 and CD8 antibodies would improve bone formation following intramuscular injections of human BMP-4 and BMP-9 adenoviral vectors (ADhBMP4 and ADhBMP9) in Sprague-Dawley rats. Cs. A was injected into the thigh muscle. After 2 days, ADhBMP4, ADhBMP9, and the antibodies were separately injected into the left and right rear legs. At this time, the number of CD4+/CD3+ cells was significantly lower and the number of CD8+/CD3+ cells higher in the Cs. A group than in the control group (Po0.01). The total number of white blood cells 3 days following injection of CD4 and CD8 antibodies was significantly lower than that before the injection (Po0.01). At 4 weeks after the viral and antibody injections, mean bone volumes at the ADhBMP9 treatment sites were 0.2970.01 cm 3 in the viral control group, 0.1770.03 cm 3 in the Cs. A-ADhBMPs group, and 0.5970.07 cm 3 in the antibodies-ADhBMPs group. ADhBMP4 did not induce new bone formation in any group. This study demonstrates that local immunomodulation may improve the osteogenic potential of bone morphogenetic protein gene therapy in the clinical setting.
PubMed, May 4, 1995
The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIr) is required for the a... more The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIr) is required for the activation of transforming growth factor beta, and previously we have found its expression to be significantly reduced in both rat and human hepatocellular carcinomas (HCCs). Therefore, we have postulated that loss of the M6P/IGFIIr gene may be mechanistically involved in liver carcinogenesis. Using the polymerase chain reaction, we utilized two polymorphisms in the 3' untranslated region of the M6P/IGFIIr gene to screen non-cirrhotic, hepatitis virus negative patients with hepatocellular tumors for LOH. Twenty-two of 36 (61%) patients were informative (heterozygous), and 14/22 (64%) liver tumors had LOH; 11/16 (69%) carcinomas, 1/3 (33%) fibrolamellar tumors and 2/3 (67%) adenomas. This is the first report of LOH at the M6P/IGFIIr locus in human hepatocellular tumors, and the presence of LOH in adenomas suggests that allelic loss may be an early event in the etiology of HCCs. These results support the hypothesis that the M6P/IGFIIr gene may function as a tumor suppressor gene in the liver.
Interventional Neuroradiology, Mar 1, 1998
We determined the propensity for and reversibility of transforming growth factor-f3 (TGFf3) bindi... more We determined the propensity for and reversibility of transforming growth factor-f3 (TGFf3) binding to uncoated Guglielmi Detachable Coils (GDC) and to GDC coated with extracellular matrix (ECM) proteins. Three 1.0 centimetre samples each of uncoated GDC-18 and of GDC-18 coated with either poly-L-lysine, laminin, type I collagen, type IV collagen, fibronectin, or poly-L-lysine and laminin were prepared. These samples were immersed briefly in a solution containing [125_ labelled TGFf3 at a concentration of 0.225 pg/ml with initial specific activity of 123.3 mCilmg (DuPont-NEN, Billerica, MA), and were counted using a scintillation counter. Each sample was then placed in a vial containing saline, shaken for 60 seconds, and counted again. Selected samples were immersed for varying periods within the TGFf3 solution and counted before and after saline rinse. Samples were rinsed one week after initial rinsing and counted again. The amount of binding between coil types was compared using the Student t test. For all samples initial binding ofTGFf3 was in the order of 60-120 pg!cm. For the pre-rinse data there were no statistically significant differences between the amount bound to any single coil coating type relative to other coatings. Compared to the initial accumulations, the amount remaining after rinsing ranged from 40% (poly-L-lysine) to 63% (poly-L-lysine with laminin), with a mean of 55% among the seven coil types. After rinsing there was more growth factor remaining on uncoated coils than on poly-L-lysine-coated coils (p=0.05), fibronectin-coated coils (p=O.Ol), and type IV collagen-coated coils (p=O.04). There was a trend toward greater residual growth factor on coils coated with poly-L-lysine and laminin compared to coils coated with poly-L-lysine alone (p=O.lO). Delayed, second rinsing of the samples one week after initial testing demonstrated only minor incremental loss of TGFf3 from the coil surfaces. After five minutes of immersion, accumulation was approximately 200% greater than that noted with brief submersion, but immersions lasting over five minutes did not yield increasing levels ofTGFf3 binding. TGFf3 binds to GDC coils. Binding is not improved with ECM protein-coated coils compared to uncoated coils. The absolute amount of TGFf3 bound to the coil will likely result in local concentrations of growth factor in the order of those required for biological activity in vivo.
Genetics, Oct 1, 1995
We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis... more We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.
Neurosurgery, Apr 1, 2003
There are anatomical and functional differences between human dental pulp (DP) and periodontal li... more There are anatomical and functional differences between human dental pulp (DP) and periodontal ligament (PDL). However, the molecular biological differences and function of these tissues are poorly understood. In the present study, we employed a cDNA microarray array to screen for differentially expressed genes (DEGs) between human DP and PDL tissues, and used the online software WebGestalt to perform the functional analysis of the DEGs. In addition, the STRING database and KEGG pathway analysis were applied for interaction network and pathway analysis of the DEGs. DP and PDL samples were obtained from permanent premolars (n=16) extracted for orthodontic purposes. The results of the microarray assay were confirmed by RT-qPCR. The DEGs were found to be significantly associated with the extracellular matrix and focal adhesion. A total of 10 genes were selected to confirm the results. The mRNA levels of integrin alpha 4 (ITGA4), integrin alpha 8 (ITGA8), neurexin 1 (NRXN1) and contactin 1 (CNTN1) were significantly higher in the DP than in the PDL tissues. However, the levels of collagen type XI alpha 1 (COL11A1), aggrecan (ACAN), collagen type VI alpha 1 (COL6A1), chondroadherin (CHAD), laminin gamma 2 (LAMC2) and laminin alpha 3 (LAMA3) were higher in the PDL than in the DP samples. The gene expression profiles provide novel insight into the characterization of DP and PDL tissues, and contribute to our understanding of the potential molecular mechanisms of dental tissue mineralization and regeneration.
The Journal of Immunology
Oil from Nigella sativa seeds (“black seed oil”) has been used for health care and medical purpos... more Oil from Nigella sativa seeds (“black seed oil”) has been used for health care and medical purposes for over 2000 years. The major active compound thymoquinone (TQ) has been shown to have immune regulatory effects in various diseases. Tumor microenvironment is critical for tumor development. Immune cells, particularly T cells, are central players to kill tumor cells and affect tumor progression and metastasis. The impact of TQ on human T cell-mediated anti-tumor response was investigated. Using human PBMCs, we activated T cells with anti-CD3/CD28 in vitro, and treated cells with TQ. First, we tested the impact of TQ on T cell activation, proliferation and apoptosis; second, the anti-tumor effect of T cells after treatment with TQ; last, we determined alterations in T cells gene expression at the RNA level after TQ treatment. TQ treatment increased activation and proliferation of T cells at days 2–4 and more activation-induced cell death at day 3. After 7–11 days, TQ-treated cells sh...
Radiology, 1998
To demonstrate in vivo that platinum embolic coils can be used to deliver genetically modified, g... more To demonstrate in vivo that platinum embolic coils can be used to deliver genetically modified, growth factor-secreting fibroblast grafts into the endovascular space with the long-term goal of improving fibrosis within coil-embolized cerebral aneurysms. Murine fibroblasts that contained multiple inserts of the DNA for human basic fibroblast growth factor were grown in culture onto 10-mm-long segments of Guglielmi detachable coils. Control (n = 4) and fibroblast-bearing (n = 4) coils were implanted into the common carotid artery in nude rats. The arterial segments that contained the coil were harvested after 14 or 35 days. Cellular content and collagen formation in the treated vessels were assessed histologically. At both 14 and 35 days, samples with control coils showed primarily involuting blood elements with minimal fibroblast proliferation or collagen formation. At 14 days, samples with fibroblast-bearing coils showed extensive fibroblast proliferation. At 35 days, samples with fibroblast-bearing coils showed marked interval fibroblast proliferation and collagen formation. Platinum coils can be used as a cell delivery device. Direct intravascular implantation of growth factor-secreting fibroblast grafts leads to improved intravascular scar formation, therefore theoretically reducing the potential for aneurysm regrowth or coil compaction.
Radiology, 1998
To evaluate the growth and adhesion characteristics in vitro of genetically modified, basic fibro... more To evaluate the growth and adhesion characteristics in vitro of genetically modified, basic fibroblast growth factor-producing fibroblasts on platinum detachable coils. Coils of two sizes were coated with laminin, poly-L-lysine, fibronectin, and type I and type IV collagen and were cultured with basic fibroblast growth factor-secreting fibroblasts. Type I collagen strands were inserted in the lumen of some coils. Cellular proliferation and adherence during passage of coils through microcatheters were studied with both light and scanning electron microscopy. Growth factor concentration in the culture medium was measured. Rapid cellular proliferation was noted on all coated coils except those coated with type IV collagen. Proliferation on uncoated coils was slightly slower than on most coated coils, although confluent cell layers were present on uncoated larger-diameter coils within 48 hours. Cells had a marked propensity to grow between the primary coil windings into the coil lumen, except in coils that contained collagen filaments. Passage through microcatheters caused widespread stripping of cells from the outer surface of coils, especially the uncoated samples. Viable cells remained in the coil lumen. Supernatant contained high concentrations of growth factor. Platinum embolic coils are a promising mechanism of cell delivery for stimulation of scar formation or other desirable biologic effects.
Proceedings of the West Virginia Academy of Science, Jul 26, 2016
Response of glioblastoma cells to activation of the G-protein coupled estrogen receptor, GPER1/GP... more Response of glioblastoma cells to activation of the G-protein coupled estrogen receptor, GPER1/GPR30, and possible crosstalk with the aryl hydrocarbon receptor. Glioblastomas are almost invariably fatal brain tumors. Glioblastoma incidence in men is 1.5 times that of pre-menopausal women and this difference decreases postmenopause. Therefore estrogen is thought to play a protective role, although the mechanism is not well understood. Glioblastomas produce and respond to a tryptophan derivative, kynurenine, that binds and activates the aryl hydrocarbon receptor (AHR). Promiscuity of ligand binding can result in cross talk between AHR and nuclear estrogen receptors and both receptor types are known to use the p300 acetyltransferase as a cofactor. We demonstrated that inhibition of p300 resulted in a reduction of proliferation of CH157-MN meningioma cells. Treatment with βestradiol or kynurenine partially abrogated this effect, suggesting both ligands may act through another receptor that we hypothesize to be the G-protein linked estrogen receptor, GPER1/GPR30. We verified the expression of GPER1, estrogen receptor β; some estrogen receptor β isoforms and AHR by CH157-MN cells and U-87 and A-172 glioblastoma cells. Inhibition of p300 with C646 reduced proliferation of both U87 and A172 cells. In U87 cells, this effect was abrogated by co-treatment with βestradiol, the xenobiotic AHR agonist 2',3',7,8'-tetrachlorodibenzo-p-dioxin, and the GPER1 agonist G-1. Exogenous kynurenine did not abrogate the effect of p300 inhibition. However, in A172 cells, none of the agonists abrogated the effect of p300 inhibition, possibly due to the higher kynurenine production in A172 cells. Treatment of A172 and U87 cells with G-1 resulted in decreased cell proliferation which was abrogated by co-treatment with the GPER1 antagonist G-36.
<p>Different controls with different levels of ethanol, the solvent used for dissolving cap... more <p>Different controls with different levels of ethanol, the solvent used for dissolving capsaicinoids in the study, were tested at 24 and 72 H. Control-1 contains culture media without ethanol. All other controls contain culture media with different levels of ethanol. Control-2, -3, -4 and -5 contained 0.2, 0.4, 0.6 and 0.8% ethanol, respectively. These were the corresponding controls for 100, 200, 300 and 400 μM capsaicin as well as dihydrocapsaicin treatments; and 0.75, 1.50, 2.25 and 3.00 g L<sup>-1</sup> ghost pepper treatments, respectively. Control-6, -7, -8 and -9 contained 0.24, 0.48, 0.72 and 0.96% ethanol, respectively. These were the corresponding controls for 100 + 20; 200 + 40; 300 + 60; and 400 + 80 μM capsaicin + dihydrocapsaicin treatments, respectively (refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206183#pone.0206183.g002" target="_blank">Fig 2</a> for treatments). Values are mean ± SD; n = 4; NS = Not significantly different from control-1 (<i>p</i> ≤ 0.05).</p
Molecular Therapy, 2004
Although adenoviral vectors have a very broad range and high efficiency of cell transduction both... more Although adenoviral vectors have a very broad range and high efficiency of cell transduction both in vitro and in vivo, the uses of adenoviral vectors have been significantly limited in immunocompetent animals. We tested whether the amount of protein expression was the major factor determining the foreign gene functions. The recombinant adenoviral vectors of human bone morphogentic protein 4 and luciferase were used to answer this question. Direct viral injection and ex vivo methods were chosen in immunodefective athymic nude (AN) and immunocompetent SD rats. On Day 0, the primary rat bone marrow cells were infected with either ADhBMP4 or ADLUC at 107 PFU/flask or PBS. At the same time, five AN and SD rats were given a direct injection of either ADhBMP4 or ADLUC (107 PFU/100 μl) into the right thigh. On Day 1, both control and virally infected cells were injected into the left thigh with 2 × 106 cells/100 μl/flask. The rats underwent imaging performed with a cooled CCD before and after an intraperitoneal luciferin injection on Days 1 and 7. On Day 1, the densities of luminescent signals (×107 photons/sec) were the following: 1.83 ± 0.56 for SD rats with direct ADLUC injection; 3.73 ± 1.15 for SD rats with ex vivo ADLUC injection; 5.01 ± 1.91 for AN rats with direct ADLUC injection; and 11.9 ± 3.35 for AN rats with ex vivo ADLUC injection. The luminescent signals in the control groups were the same as background. By Day 7, the percentages of luminescence densities in ADLUC groups (compared with the same site on Day 1) were the following: 14.1% for SD rats with direct ADLUC injection; 1.06% for SD rats with ex vivo ADLUC injection; 25.4% for AN rats with direct ADLUC injection; and 0.8% for nude rats with ex vivo ADLUC injection. The animals were scanned with CT 1 month after injection. The bone volumes (cm3) of ADhBMP4 induced were 0.85 ± 0.08 for direct injection and 0.15 ± 0.03 for ex vivo in AN rats, and no bone for direct injection and 0.12 ± 0.02 for ex vivo in SD rats. No bone was observed at sites receiving injections of control cells or ADLUC. These data demonstrate that AN and SD rats display similar expression profiles of foreign gene expression delivered with adenoviral vectors, regardless of whether direct viral injection or the ex vivo method is used. As we know the amount of new bone is dose dependent in AN rats, and the average amount of bone induced by hBMP4 is smaller when the ex vivo method is employed than when direct viral injection is used. This result reflects the lower level of hBMP4 expression using the ex vivo method than directly viral injection. However, in SD rats, although hBMP4 expression may be lower after the ex vivo injection than after directly viral injection, the ex vivo ADhBMP4 delivery induces bone formation. These results indicate that the amount of BMP expression is not the major factor affecting the amount of new bone in SD rats. The major factors affecting the osteogenic potentials of ADhBMPs in SD rats may be some immune factors, which may have higher titer in direct adenoviral injection comparing to the ex vivo delivery.
Human Gene Therapy, 1999
Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standar... more Bone morphogenetic proteins (BMPs) are polypeptides that induce ectopic bone formation in standard rat in vivo assay systems. Previous studies have demonstrated the clinical utility of these proteins in spinal fusion, fracture healing, and prosthetic joint stabilization. Gene therapy is also a theoretically attractive technique to express BMPs clinically, since long-term, regulatable gene expression and systemic delivery with tissue-specific expression may be possible in future. This study was performed to determine whether an adenoviral vector containing the BMP-2 gene can be used to express BMP-2 in vitro and promote endochondral bone formation in vivo. In vitro, U87 MG cells transduced per cell with 20 MOI of an adenoviral construct containing the BMP-2 gene under the control of the universal CMV promoter (Ad-BMP-2) showed positive antibody staining for the BMP-2 protein at posttransfection day 2. The synthesis and secretion of active BMP-2 into the conditioned medium of Ad-BMP-2-transduced 293 cells were confirmed by Western blot analysis and the induction of alkaline phosphatase activity in a W-20 stromal cell assay. In vivo, Sprague-Dawley rats and athym ic nude rats were injected with Ad-BMP-2 in the thigh musculature and were sacrificed on day 3, 6, 9, 12, 16, 21, 60, and 110 for histological analysis. The Sprague-Dawley rats showed evidence of acute inflammation, without ectopic bone formation, at the injection sites. In the athymic nude rats, BMP-2 gene therapy induced mesenchymal stem cell chemotaxis and proliferation, with subsequent differentiation to chondrocytes. The chondrocytes secreted a cartilaginous matrix, which then mineralized and was replaced by mature bone. This study demonstrates that a BMP-2 adenoviral vector can be utilized to produce BMP-2 by striated muscle cells in athymic nude rats, leading to endochondral bone formation. However, in immunocompetent animals the endochond ral response is attenuated, secondary to the massive immune response elicited by the firstgeneration adenoviral construct.
Acta Neuropathologica, 2002
Tissue Engineering, 2006
The osteogenic potential of AAV5hBMP6 was compared with that of ADhBMP6 in immunodeficient and im... more The osteogenic potential of AAV5hBMP6 was compared with that of ADhBMP6 in immunodeficient and immunocompetent rats. AAV5hBMP6 (2.3 x 10(12) particles) and ADhBMP6 (5 x 10(7) PFU) elicited viral antibody production in immunocompetent rats. Among rats that received AAV5hBMP6, the earliest time points at which the bone was visible under CT scanner were 30 days in 2-month-old Sprague-Dawley (SD) rats and 60 days in 18-month-old SD rats. The mean volumes of ectopic bone 90 days after viral injection were 0.31 +/- 0.14 cm(3) in athymic nude rats, 0.64 +/- 0.12 cm(3) in 2-month-old SD rats, and 0.21 +/- 0.10 cm(3) in 18-month-old SD rats. In contrast, among rats that received ADhBMP6, the earliest time points to observe the bone formation by CT scan were 15 days in 2-month-old rats and no bone formation in 18-month-old SD rats. The mean volumes of ectopic bone were 4.17 +/- 0.05 cm(3) in athymic nude rats and 0.06 +/- 0.03 cm(3) in 2-month-old SD rats. Although both types of viruses induced an immune response in immunocompetent animals, this response played different roles in the process of bone formation induced by the BMP6 vectors.
Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Produ... more Pepper (Capsicum annuum L.) is an economically important crop with added nutritional value. Production of capsaicin is an important quantitative trait with high environmental variance, so the development of markers regulating capsaicinoid accumulation is important for pepper breeding programs. In this study, we performed association mapping at the gene level to identify single nucleotide polymorphisms (SNPs) associated with capsaicin pathway metabolites in a diverse Capsicum annuum collection during two seasons. The genes Pun1, CCR, KAS and HCT were sequenced and matched with the whole-genome sequence draft of pepper to identify SNP locations and for further characterization. The identified SNPs for each gene underwent candidate gene association mapping. Association mapping results revealed Pun1 as a key regulator of major metabolites in the capsaicin pathway mainly affecting capsaicinoids and precursors for acyl moieties of capsaicinoids. Six different SNPs in the promoter sequence...
Glioma incidence in males is 1.5 times that of females, thus it is suspected that female sex horm... more Glioma incidence in males is 1.5 times that of females, thus it is suspected that female sex hormones play a protective role against gliomas. Compounds found in water contaminated by extractive industry waste can disrupt hormone mediated signaling. Studies have been done on endocrine disrupting effects on estrogen signaling, but little has been done on their effects on progesterone signaling. Additionally, the focus has been on classical receptors, with almost no investigation of G-protein coupled receptors. Here we investigate the effects of exposure to coal dust on cell proliferation and expression of progestin and adipoQ receptors (PAQRs) in two human glioma cell lines, A172 and U87. Treatments with coal dust and treatments with progesterone were performed. End point RT-PCR demonstrated that A172 cells express PAQR3, PAQR5, and PAQR9. This was the first demonstration of PAQR5 expression in A172 cells. U87 cells express PAQR3, PAQR5, PAQR7, PAQR8, and PAQR9. Real time RT-PCR was p...