Human Tissue Plasminogen Activator Gene Research Articles

Association between Alu insertion/deletion polymorphism in intron 8 of human tissue plasminogen activator gene (PLAT) and risk of age-related macular degeneration

Background/Aim: Age-related macular degeneration (AMD) is major reason of blindness in human. Plasminogen is converted to plasmin by tissue plasminogen activator protein (PLAT, formerly known as TPA). A polymorphism in intron 8 of PLAT gene has been reported, either with (insertion) or without (deletion) a 311 bp Alu sequence. This polymorphism is associated with plasma levels of glycoprotein t-PA. t-PA is expressed in the retina and is involved in the development of the eye. It can be hypothesised that the PLAT polymorphism may be correlated with AMD. Therefore, the current study was conducted. Methods: A total of 121 AMD patients and 108 healthy subjects were included in the study. Genotyping was performed by PCR. The strength of the association between AMD and polymorphism was expressed by estimating the odds ratio (OR). Results: There was a significant relationship between the Del/Del genotype and susceptibility to AMD (OR = 2.25, 95 % CI = 1.07-4.69, p = 0.031). After adjusting for various factors such as age, smoking habit and workplace, a similar relationship was obtained (OR = 2.51, 95 % CI = 1.01-6.23, p = 0.049). Conclusions: The homozygosity of the Del allele was found to increase the susceptibility to AMD. This polymorphism may contribute to the risk of AMD in population.

Alu DNA Polymorphism of Human Tissue Plasminogen Activator (tPA) Gene in Diabetic Jordanian Patients Patients

Background:Hypercoagulability and hypofibrinolysis are among the symptoms exhibited by diabetic patients. Our study aimed to address the polymorphic nature of Alu DNA fragment in the human tissue plasminogen activator gene within diabetes mellitus (DM) Jordanian patients.Methods:Genomic DNA was isolated from 76 DM patients and 60 non-diabetic Jordanian individuals, and the Alu fragment was amplified using PCR. Results:The results showed that 80% of the non-diabetic Jordanian subjects were homozygotes for the deletion of the Alu fragment (Alu-/-), 16.7% were homozygotes for its insertion (Alu+/+), and 3.3% were heterozygotes (Alu+/-). Besides, 36.8% of the diabetic patients exhibited the Alu-/- or Alu+/- genotype, and 26.3% were Alu+/+. The Alu-/- genotype occurred less frequently in the diabetic individuals. Conclusion:The high frequency of the Alu-/- genotype constitutes a protective deletion with respect to DM within the normal subjects.

ChAdOx1 and MVA based vaccine candidates against MERS-CoV elicit neutralising antibodies and cellular immune responses in mice

The Middle East respiratory syndrome coronavirus (MERS-CoV) has infected more than 1900 humans, since 2012. The syndrome ranges from asymptomatic and mild cases to severe pneumonia and death. The virus is believed to be circulating in dromedary camels without notable symptoms since the 1980s. Therefore, dromedary camels are considered the only animal source of infection. Neither antiviral drugs nor vaccines are approved for veterinary or medical use despite active research on this area. Here, we developed four vaccine candidates against MERS-CoV based on ChAdOx1 and MVA viral vectors, two candidates per vector. All vaccines contained the full-length spike gene of MERS-CoV; ChAdOx1 MERS vaccines were produced with or without the leader sequence of the human tissue plasminogen activator gene (tPA) where MVA MERS vaccines were produced with tPA, but either the mH5 or F11 promoter driving expression of the spike gene. All vaccine candidates were evaluated in a mouse model in prime only or prime-boost regimens. ChAdOx1 MERS with tPA induced higher neutralising antibodies than ChAdOx1 MERS without tPA. A single dose of ChAdOx1 MERS with tPA elicited cellular immune responses as well as neutralising antibodies that were boosted to a significantly higher level by MVA MERS. The humoral immunogenicity of a single dose of ChAdOx1 MERS with tPA was equivalent to two doses of MVA MERS (also with tPA). MVA MERS with mH5 or F11 promoter induced similar antibody levels; however, F11 promoter enhanced the cellular immunogenicity of MVA MERS to significantly higher magnitudes. In conclusion, our study showed that MERS-CoV vaccine candidates could be optimized by utilising different viral vectors, various genetic designs of the vectors, or different regimens to increase immunogenicity. ChAdOx1 and MVA vectored vaccines have been safely evaluated in camels and humans and these MERS vaccine candidates should now be tested in camels and in clinical trials.

The tissue plasminogen activator gene promoter: a novel tool for radiogenic gene therapy of the prostate?

Radiation therapy is a treatment modality routinely used in cancer management so it is not unexpected that radiation-inducible promoters have emerged as an attractive tool for controlled gene therapy. The human tissue plasminogen activator gene promoter (t-PA) has been proposed as a candidate for radiogenic gene therapy, but has not been exploited to date. The purpose of this study was to evaluate the potential of this promoter to drive the expression of a reporter gene, the green fluorescent protein (GFP), in response to radiation exposure. To investigate whether the promoter could be used for prostate cancer gene therapy, we initially transfected normal and malignant prostate cells. We then transfected HMEC-1 endothelial cells and ex vivo rat tail artery and monitored GFP levels using Western blotting following the delivery of single doses of ionizing radiation (2, 4, 6 Gy) to test whether the promoter could be used for vascular targeted gene therapy. The t-PA promoter induced GFP expression up to 6-fold in all cell types tested in response to radiation doses within the clinical range. These results suggest that the t-PA promoter may be incorporated into gene therapy strategies driving therapeutic transgenes in conjunction with radiation therapy.

Identification of the UV-Responsive Sequence in the Human Tissue Plasminogen Activator Gene

Functional analysis of regulatory elements in the human tissue plasminogen activator (tPA) gene showed that its promoter (-119/+169) is activated by UV irradiation in HeLa cells. We demonstrated here that the AP-2 like CCCCACCC sequence is involved in the UV-mediated activation and Sp1 binds to the sequence.

Detection of the GSTM1*0 allele by long polymerase chain reaction

Approximately 50% of individuals in most human populations completely lack activity of the detoxifying enzyme glutathione S-transferase M1. The medical consequences of this deficiency have been extensively investigated in molecular epidemiological studies, but possible differences of the highly active homozygous genotype versus the moderately active heterozygous genotype could not be considered because many currently used polymerase chain reaction (PCR) assays cannot distinguish the homozygous genotypes GSTM1 *A/A and GSTM1*B/B from the heterozygous genotypes GSTM1*A/0 and GSTM1*B/0. Here, we describe that long PCR is suitable for this purpose by specifically producing a signal for the GSTM1*0 allele. Based on the published cluster of GSTM genes (GSTM1 to -M5), a 13-kb segment spanning the site of the GSTM1 deletion was amplified using a GSTM2-specific forward primer (5'-CATCGCTTATGATGTCCTTGAGAGAAACCAAG-3') and a reverse primer, which is specific for the upstream region of GSTMS (5'-GCGTTTCTGAGGACTGGACTGATGATCG-3'). Any failure in the amplification was controlled by co-amplification of a 15-kb human tissue plasminogen activator gene fragment. The GSTM1*0-specific 13-kb amplicon appears in all carriers of one or two null alleles, but in none of the 14 tested GST1*A/B samples which served as controls since individuals with this genotype are known to lack any GSTM1*0 allele. While conventional PCR assays for detection of the GSTM1 deletion differentiated homozygously deficient from hetero- or homozygously active individuals, this long PCR assay differentiates homozygously active from hetero- or homozygously deficient individuals. Using both assays, an unambiguous differentiation into carriers of zero, one or two active alleles of GSTM1 is possible.

Retroviral vector-mediated transfer and expression of human tissue plasminogen activator gene in human endothelial and vascular smooth muscle cells.

Enhancement of the fibrinolytic activity of vascular cells by tissue plasminogen activator (tPA) gene transfer has considerable clinical potential. However, it is unknown whether greater constitutive expression of the tPA gene might increase plasminogen activator inhibitor-1 (PAI-1) secretion, which could negate expected increases in fibrinolytic activity that accompany greater tPA protein production. The objective of this investigation was to determine whether transduction of human endothelial cells (EC) and vascular smooth muscle cells (SMC) with a retroviral vector containing the human tPA gene would increase tPA production and what effect this would have on endogenous PAI-1 secretion and subsequent fibrinolytic activity. Cultivated human EC and SMC either were transduced with a murine leukemia retroviral vector (MFG) containing the human tPA gene and, in the case of controls, the lacZ gene, or they were exposed to media alone. On days 14 and 28 after transduction, supernatent tPA antigen and PAI-1 antigen levels were measured by ELISA, and supernatent tPA activity was quantitated with a spectrolyse tPA/PAI assay. Southern and Northern blot analyses documented integration and transcription of the tPA gene in both EC and SMC. Greater tPA antigen production occurred in MFG-tPA-transduced EC and SMC compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). The tPA activity increased in transduced human saphenous vein EC (up to 5.1-fold) and human iliac artery EC (up to 4.7-fold), but no increased tPA activity occurred in transduced SMC, compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). PAI-1 antigen was unchanged in transduced SMC but decreased in MFG-tPA-transduced EC (p < 0.05). PAI-1 mRNA was unchanged in the transduced EC and SMC compared with nontransduced cells, suggesting that posttranslational events may have caused the changes in EC PAI-1. This investigation demonstrated that MFG-mediated tPA gene transfer into human EC resulted in a significant increase in tPA activity. Enhancement of adult human EC fibrinolytic activity by transfer of the human tPA gene has not been previously reported and represents a necessary finding in the development of this gene therapy technology for the prevention of thrombotic complications of vascular disease.

TATA box-independent transcription of the human tissue plasminogen activator gene initiates within a sequence conserved in related genes

Transcription of the human tissue-type plasminogen activator (tPA) gene has been reported to initiate from a single site proximal to a TATA box motif (1985, J. Biol. Chem. 260, 11223–11230). In this study, we utilized primer extension analysis to evaluate the tPA mRNA start site in phorbol-12-myristate 13-acetate (PMA) induced WI-38 man lung fibroblast cells. Whilst some tPA mRNA initiated from the predicted TATA-proximal location (+1), a 10-fold greater proportion of tPA mRNA transcripts initiated 110 bases downstream from a sequence conserved and utilized as the TATA-independent transcription start site in the rodent tPA genes. Moreover, the transfection and expression in different cell types of a cosmid containing the entire human tPA gene resulted in utilization of the same downstream (+110) start site. We propose that this, rather than the previously published position, is the major transcriptional initiation point for the human tPA gene. A core sequence (5′-CAGAGCTG-3′) was identified which is common to the TATA-independent mRNA start sites of the human, mouse and rat tPA genes, and which demonstrates only partial similarity to sequences found at the initiation point of other TATA-independent genes.

A polymorphic dinucleotide repeat in intron 1 of the human tissue plasminogen activator gene.

Journal Article A polymorphic dinucleotide repeat in intron 1 of the human tissue plasminogen activator gene Get access W. Thomas, W. Thomas Department of Molecular Biology, Genentech IncSouth San Francisco, CA 94080, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar D. Drayna D. Drayna Department of Molecular Biology, Genentech IncSouth San Francisco, CA 94080, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Human Molecular Genetics, Volume 1, Issue 2, May 1992, Page 138, https://doi.org/10.1093/hmg/1.2.138-a Published: 01 May 1992

Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a 32P-labeled oligomer. After 16 h, the signal was 12 times greater than the 32P signal. The detection of the single-copy tissue plasminogen activator gene in 0.25 μg of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.

Dinuclotide repeat polymorphism at the human tissue plasminogen activator gene (PLAT)

Dinuclotide repeat polymorphism at the human tissue plasminogen activator gene (PLAT)

Transcription efficiency along the tissue-plasminogen-activator cDNA gene in Escherichia coli

Transcription along the human tissue-plasminogen activator gene ( tpa) cloned in Escherichia coli was studied by inserting the 4.5S RNA gene at various locations within tpa, and measuring the accumulation of 4.5S RNA. A six-fold decrease in transcription of 4.5S RNA was observed as the distance from the Ptrc promoter to the 4.5 S RNA gene increased. Independent measurements indicated that the quantity of initiations from the Ptrc promoter could not account for the differences observed. Because the 4.5S RNA is stable, the decrease in transcription was assumed to be due to premature transcription-termination within the tpa gene. This polar effect could contribute to poor expression of tpa in E. coli.

The human tissue plasminogen activator gene.

The nucleotide sequence of the human tissue plasminogen activator (t-PA) gene has been established. A total of 36,594 base pairs (bp) was sequenced; this included 32,720 bp from the site of initiation of transcription to the polyadenylation site, in addition to 3,530 and 344 bp of 5' and 3' flanking DNA, respectively. Thirteen intervening sequences divide the gene into 14 coding regions; the size range for exons is 43-914 bp, while that for introns is 111-14,257 bp. The gene and 5' flanking region contain 28 copies of Alu repetitive DNA and a single KpnI repeat. The transcription initiation site was identified by S1 nuclease, exonuclease VII, and primer extension analysis as an A residue; "TATA" and "CAAT" boxes are located in the expected positions upstream of this proposed site. Results of the analysis of the gene sequence and its comparison with data banks are described. The protein and gene structures of tissue and urokinase plasminogen activator are compared; based on these features the evolutionary relationship of the two human plasminogen activators appears to be close.

Tissue-type plasminogen activator gene is on chromosome 8.

Tissue plasminogen activator is one of the two plasminogen activators, both serine proteases, that catalyze the conversion of inactive plasminogen to plasmin, which then degrades the fibrin network of blood clots. By combining somatic cell genetics, in situ hybridization, and Southern blot hybridization, we localized the human tissue plasminogen activator gene to the pericentromeric region of chromosome 8.