Baseline bio-accumulation concentrations and resulting oxidative stress in Synodontis zambezensis after an acute laboratory exposure to 4,4'-DDT.

The use of 1,1'-(2,2,2-Trichloro-1,1-ethanediyl)bis(4-chlorobenzene) (DDT) as a pesticide for the control of insects vectors responsible for the spread of many life threatening diseases was officially banned in 1972 by the United States Environmental Protection Agency (USEPA). It was banned throughout the world, in most developed countries, because of the toxic effects it causes in wildlife, including birds and fish. However, DDT is still used in approximately 43 African countries, including South Africa, to control the spread of malaria. The lipophilic nature of DDT and therefore its persistence in the environment makes it extremely important for laboratory based studies to be conducted in an effort to evaluate the accumulation potential and possible physiological effects of DDT in aquatic organisms under controlled conditions. The aim of this study was to establish baseline bioaccumulation concentrations within Synodontis zambezensis following an acute exposure to 4,4'-DDT. The three metabolites analysed were 4,4'-DDE, 4,4'-DDD and 4,4'-DDT. None of the 2,4'-isomers were analysed in this study since the acute exposure used a solution of 98.7% pure 4,4'-DDT (Sigma-Aldrich PESTANAL®, Analytical Standard, CAS-No 50-29-3, Batch number SZBE057XV) and not a mixture of 4,4'-DDT and 2,4'-DDT as found in technical grade DDT. Soxhlet extraction of tissue samples and liquid/liquid extraction of water samples followed by analysis through Gas-chromatography mass-spectrophotometry was completed. Mean 4,4'-DDE, 4,4'-DDD and 4,4'-DDT concentrations ranged from 15.34 ng/g to 45.34 ng/g, 28.16 ng/g to 63.25 ng/g and 28.64 ng/g to 96.21 ng/g respectively. All of the accumulated concentrations fell within environmentally relevant concentrations with no input through the food web. The accumulated concentrations of 4,4'-DDT and its three metabolites resulted in oxidative stress responses within the gills and the liver tissue of S. zambezensis. Significant differences (p ≤ .05) were observed between malondialdehyde (MDA) and reduced glutathione (GSH) within the liver and in superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) in the gills.

Biomarkers in tigerfish (Hydrocynus vittatus) as indicators of metal and organic pollution in ecologically sensitive subtropical rivers.

Studies have shown high levels of contamination of both metals and organochlorine pesticides (OCPs) in aquatic systems of the world renowned Kruger National Park, South Africa. With effects evident in top predators, including, unexplained Crocodylus niloticus deaths and organ level and histological changes in Hydrocynus vittatus. A suite of biomarkers reflecting exposure and were selected to evaluate biological responses of H. vittatus to anthropogenic stressors as well as to evaluate whether the chosen suite of biomarkers could successfully distinguish between the different pollution profiles present in the selected rivers. During this study a clear relationship was found between exposure to environmental contaminants and the concomitant responses of H. vittatus to these stressors. The ensuing biomarker responses indicated that there is a physiological attempt to deal with, and mitigate the deleterious effects that metals and OCPs may induce. In the Luvuvhu River there is a clear indication in H. vittatus of the stimulation of anti-oxidant protective mechanisms in response to internal OCP exposure. This is reflected by the increasing cytochrome P-450, superoxide dismutase, and more specifically reduced glutathione, which resulted in decreased lipid and protein breakdown (reflected in decreased lipid peroxidation and protein carbonyl levels). Consequently H. vittatus populations of the Luvuvhu River are under greater cumulative stress and this is reflected in the lower energy budgets. Our results further show the integrated application value of the current suite of biomarkers in assessing responses of subtropical fish to metal and OCP exposure as the entire suite of biomarkers when used in conjunction were able to explain 100% of the variation in the data.

Human Health Risk from Consumption of Marine Fish Contaminated with DDT and Its Metabolites in Maputo Bay, Mozambique.

Many countries with incidence of malaria, including those surrounding Maputo Bay, use dichloro-diphenyl-trichloroethane (DDT) to reduce mosquitoes. This study is the first to estimate the human health risk associated with consumption of marine fish from Maputo Bay contaminated with DDTs. The median for ∑DDTs was 3.8 ng/g ww (maximum 280.9 ng/g ww). The overall hazard ratio for samples was 1.5 at the 75th percentile concentration and 28.2 at the 95th percentile. These calculations show increased potential cancer risks due to contamination by DDTs, data which will help policy makers perform a risk-benefit analysis of DDT use in malaria control programs in the region.

Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam.

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p-hydroxydiazepam [p-OH-DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p-OH-DZP was significantly less in the horse. Inhibition assays with a CYP-specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos-7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.

Partial cloning of CYP2C23a genes and hepatic protein expression in eight representative avian species.

Large interspecies differences in avian xenobiotic metabolism have been revealed by microsome-based studies, but specific enzyme isoforms in different bird species have not yet been compared. We have previously shown that CYP2C23 genes are the most induced CYP isoforms in chicken liver. In this study, we collected partial CYP2C23a gene sequences from eight avian species (ostrich, blue-eared pheasant, snowy owl, great-horned owl, Chilean flamingo, peregrin falcon, Humboldt penguin, and black-crowned night heron) selected to cover the whole avian lineage: Paleognathae, Galloanserae, and Neoaves. Genetic analysis showed that CYP2C23 genes of Galloanserae species (chicken and blue-eared pheasant) had unique characteristics. We found some duplicated genes (CYP2C23a and CYP2C23b) and two missing amino acid residues in Galloanserae compared to the other two lineages. The genes have lower homology than in other avian lineages, which suggests Galloanserae-specific rapid evolutionary changes. These genetic features suggested that the Galloanserae are not the most representative avian species, considering that the Neoaves comprise more than 95% of birds. Moreover, we succeeded in synthesizing an antipeptide polyclonal antibody against the region of CYP2C23 protein conserved in avians. However, comparative quantitation of CYP2C23 proteins in livers from six species showed that expression levels of these proteins differed no more than fourfold. Further study is needed to clarify the function of avian CYP2C23 proteins.

Urinary steroid profiling using liquid chromatography-tandem mass spectrometry for the diagnosis of canine Cushing's syndrome.

Serum cortisol measurements by chemiluminescence enzyme immunoassay (CLEIA) are widely used to diagnose hypercortisolism (HC) or Cushing's syndrome in dogs. However, they are associated with problems such as the need for multiple blood collections under stressful conditions or cross-reactivity between hormones. Therefore, a less invasive and more accurate diagnostic method is required. This study aimed to develop a urinary steroid profile analysis method using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and to evaluate its clinical usefulness. Sixty-five healthy dogs and 38 dogs with suspected HC were included in the study. Using LC/MS/MS, the levels of 11 steroid hormones in the urine were determined. We established the upper limit of the reference interval for each urinary steroid-to-creatinine ratio and evaluated their diagnostic performances. The levels of the five steroid hormones were significantly higher in the 14 dogs with HC than in the 24 dogs with mimicking HC and 65 healthy dogs. The urinary corticosterone-to-creatinine ratio showed the highest diagnostic accuracy (area under the curve, 0.96). A significant correlation was seen between urinary cortisol concentrations measured by LC/MS/MS and CLEIA (rs = 0.88, P <0.001), although the CLEIA measurements were significantly higher than the LC/MS/MS measurements (P <0.001). LC/MS/MS-based urinary steroid profiles are a promising tool for diagnosing canine HC.

Molecular evaluation of a new highly sensitive aryl hydrocarbon receptor in ostriches.

A 1,000-fold difference has been reported in dioxin sensitivity between avian species. This difference is because the 2 amino acids in the type 1 aryl hydrocarbon receptor (AhR1), at positions 325 and 381, correspond to Leu324 and Ser380 in chickens. The chicken had been reported to be the only avian species to possess a sensitive form of AhR1. This is the first study to reveal that the ostrich (Struthio camelus), a nonchicken species, also has a pair of amino acids (Ile-325 and Ser-381) that show high ligand affinity. However, the alignment of the AhR1 cDNA sequence showed that the AhR sequence in the ostrich was different than that of other avian species even though the critical amino acids were observed at positions 325 and 381. Ostrich AhR1 was also evaluated in a heterologous expression study. Ostrich AhR1 showed very high transcriptional activity of the cytochrome P450 1A5 (CYP1A5) gene in African Green Monkey Cercopithecus aethiops kidney cells (COS-7) treated with Sudan III. In primary cultures of ostrich kidney cells, CYP1A5 expression was induced by Sudan III at a lower (or almost identical) concentration to that observed in the chicken. The present study revealed a new AhR ligand sensitive avian species (i.e., the ostrich).

Molecular cloning and characterization of Th1 and Th2 cytokines of African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) has been implicated as the reservoir of several bovine infectious agents. However, there is insufficient information on the protective immune responses in the African buffalo, particularly in infected animals. In this study, we analysed Th1 cytokines IL-2 and IFN-γ, and Th2 cytokines IL-4 and IL-10. The cloned cDNA of IL-2, IL-4, IL-10 and IFN-γ contained an open reading frame of 468, 501, 408 and 540 nucleotides, encoding polypeptides of 155, 166, 135 and 179 amino acids, respectively. Nucleotide sequence homology of IL-2, IFN-γ and IL-4 was more than 98% between the African buffalo and cattle, which resulted in identical polypeptides. Meanwhile, IL-10 gene of African buffalo and cattle had 95% homology in nucleotide sequence, corresponding to thirteen amino acid residues substitution. Cysteine residues and potential glycosylation sites were conserved within the family Bovinae. Phylogenetic analyses including cytokines of the African buffalo placed them within a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle, water buffalo, sheep, goat, pig and artiodactyl wildlife. A deeper understanding of the structure of these cytokines will shed light on their protective role in the disease-resistant African buffalo in comparison with other closely related species.

Comparative metabolism of warfarin in rats and chickens.

Warfarin, a coumarin rodenticide, is commonly used worldwide for rodent control, and is often reported as the cause for poisoning accidents in nontarget animals, in particular bird species. However, the metabolism of warfarin in birds is still unclear. In a previous study, we found an unknown warfarin metabolite in chicken cytosolic fractions. In the present study, we aimed to clarify the cytosolic warfarin metabolites in chickens compared with those in rats. The cytosol fractions of both chicken and rat livers showed the metabolic activity of 2 diastereomers and 2 enantiomers of warfarin alcohol. In chicken cytosol, we found that the production level of (S)-warfarin-(S)-alcohol was markedly higher (32-fold) than that in rat cytosol. From the results of the inhibition assay, we finally suggest that aldehyde oxidase may mainly contribute to the warfarin alcohol products in chicken cytosol.

Prostaglandin-related immune suppression in cattle.

Prostaglandins (PGs) are lipid mediators derived from arachidonic acid by several enzymes including cyclooxygenase (COX)-1 and COX-2. We have previously shown that PGE2 regulates immune responses, such as Th1 cytokine production and T-cell proliferation, in cattle. However, it is still unclear whether other PGs are involved in the regulation of immune responses in cattle. Here, immunosuppressive profiles of PGs (PGA1, PGB2, PGD2, PGE2, PGF1α and PGF2α) were firstly examined using bovine peripheral blood mononuclear cells (PBMCs). In addition to PGE2, PGA1 significantly inhibited Th1 cytokine production from PBMCs in cattle. Further analyses focusing on PGA1 revealed that treatment with PGA1 in the presence of concanavalin A (con A) downregulated CD69, an activation marker, and IFN-γ expression in both CD4+ and CD8+ T cells. Sorted CD3+ T cells stimulated with con A were cultivated with PGA1, and IFN-γ and TNF-α concentrations decreased upon PGA1 treatment. Taken together, these results suggest that the treatment with PGA1in vitro inhibits T-cell activation, especially Th1 cytokine production, in cattle.

Evidence of impacts from DDT in pelican, cormorant, stork, and egret eggs from KwaZulu-Natal, South Africa.

DDT remains in use for malaria control in South Africa. We quantified DDTs in aquatic bird eggs from the highly biodiverse northern KwaZulu-Natal, a province of South Africa where DDT has been used for more than 80 years for malaria control. Pelican eggs had the highest ΣDDT concentration (7200 ng/g lipid mass; lm), Little Egret eggs had 6900 ΣDDT lm, African Openbill eggs had 3400 ng/g lm ΣDDT, and White-breasted Cormorant had 2400 ng/g lm. All species had non-significantly different mean concentrations of o,p'-DDT, p,p'-DDT, and ΣDDT, but with significant differences for p,p-DDE, o,p'-DDD, p,p'-DDD, %DDT, %DDD, and %lipid. The thinnest pelican eggshell (0.40 mm) had a ΣDDT concentration of 3300 ng/g lm.; the thickest shell (0.96 mm) had the lowest ΣDDT concentration at 29 ng/g lm; a 58% difference. Linear regressions of concentrations with shell thickness for the pelican eggs were significant for p,p'-DDE and p,p'-DDD, indicating risk of reproductive impairment. Compositional profiles indicate different food webs for the different species. DDT concentrations were lower than from another DDT-sprayed locality in South Africa, possible linked to differences in hydrology and rainfall. We conclude that significant ecotoxic threats associated with DDT remain in this area, and possibly threatens birds from less polluted areas. Our findings suggest continued negative human health and environmental impacts from DDT. There is an urgency to move away from DDT as quickly as possible; alternatively, to implement practices that prevent emissions of DDT to the environment while protecting human life.

Biotransport of metallic trace elements from marine to terrestrial ecosystems by seabirds.

Physical systems, such as currents and winds, have traditionally been considered responsible for transporting contaminants. Although evidence is mounting that animals play a role in this process through their movements, we still know little about how such contaminant biotransport occurs and the extent of effects at deposition sites. In the present study, we address this question by studying how rhinoceros auklets (Cerorhinca monocerata), a seabird that occurs in immense colonies (∼300 000 pairs at our study site, Teuri Island), affect contaminant levels at their colony and at nearby sites. More specifically, we hypothesize that contaminants are transported and deposited by seabirds at their colony and that these contaminants are passed on locally to the terrestrial ecosystem. To test this hypothesis, we analyzed the concentration of 9 heavy metal and metalloids, as well as δ13 C and δ15 N stable isotopes, in bird tissues, plants, and soil, both within and outside of the colony. The results show that rhinoceros auklets transport marine-derived mercury (Hg), possibly from their wintering location, and deposit Hg via their feces at their breeding site, thereby contaminating plants and soils within the breeding colony. The present study confirms not only that animals can transport contaminants from marine to terrestrial ecosystems, potentially over unexpectedly long distances, but also that bird tissues contribute locally to plant contamination. Environ Toxicol Chem 2019;38:106-114. © 2018 SETAC.

A glycomics approach to discover novel renal biomarkers in birds by administration of cisplatin and diclofenac to chickens.

Avian species have a unique renal structure and abundant blood flow into the kidneys. Although many birds die due to nephrotoxicity caused by chemicals, there are no early biomarkers for renal lesions. Uric acid level in blood, which is generally used as a renal biomarker, is altered when the kidney function is damaged by over 70%. Therefore, early biomarkers for kidney injury in birds are needed. In humans, glycomics has been at the forefront of biological and medical sciences, and glycans are used as biomarkers of diseases, such as carcinoma. In this study, a glycomics approach was used to screen for renal biomarkers in chicken. First, a chicken model of kidney damage was generated by injection of diclofenac or cisplatin, which cause acute interstitial nephritis (AIN) and acute tubular necrosis (ATN), respectively. The nephrotoxicity levels were determined by a blood chemical test and histopathological analysis. The plasma N-glycans were then analyzed to discover renal biomarkers in birds. Levels of 14 glycans increased between pre- and post administration in kidney-damaged chickens in the diclofenac group, and some of these glycans had the same presumptive composition as those in human renal carcinoma patients. Glycan levels did not change remarkably in the cisplatin group. It is possible that there are changes in glycan expression due to AIN, but they do not reflect ATN. Although further research is needed in other species of birds, glycans are potentially useful biomarkers for AIN in avian species.

Concentrations and human health risk assessment of DDT and its metabolites in free-range and commercial chicken products from KwaZulu-Natal, South Africa.

Organochlorine pesticides such as dichlorodiphenyltrichloroethane (DDT) have been used in agriculture and for disease control purposes over many decades. Reports suggest that DDT exposure may result in a number of adverse effects in humans. In the KwaZulu-Natal Province of South Africa, DDT is sprayed annually in homes (indoor residual spraying) to control the mosquito vector of malaria. In the northern part of the Province, samples of free-range chicken meat (n = 48) and eggs (n = 13), and commercially produced chicken meat (n = 6) and eggs (n = 11), were collected and analysed. Of the free-range chicken meat samples, 94% (45/48) contained DDTs (ΣDDTs median 6.1 ng/g wet weight [ww], maximum 79.1 ng/g ww). Chicken egg contents were also contaminated (ΣDDTs in free-range eggs median 9544 ng/g ww, maximum 96.666 ng/g ww; and in commercial eggs median 1.3 ng/g ww, maximum 4.6 ng/g ww). The predominant DDT congener detected was p,p'-DDE in both free-range meat (>63%) and eggs (>66%), followed by p,p'-DDT and then p,p'-DDD. Based on estimated daily intake values, calculated human risk ratio (carcinogenic) values were >1 for DDTs detected in both free-range chicken products. Consumption of free-range eggs poses a particularly high health risk.

Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases.

BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases. CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis.

The growth of any solid tumor depends on angiogenesis. Among the known angiogenic factors, vascular endothelial growth factor (VEGF) has been shown to play a pivotal role in tumor angiogenesis. However, to date, the signal transduction pathway initiated by VEGF is still not fully understood. It has been suggested that protein kinase C (PKC) plays an important role in the VEGF-induced signal transduction pathway in vitro, although the role of PKC in tumor angiogenesis in vivo still remains to be elucidated. By delivering the VEGF gene within the self-contained tetracycline-regulated retroviral vector (Retro-Tet) into hepatocellular carcinoma (HCC) cells, we manipulated VEGF expression by providing tetracycline in the drinking water to assess the tumor kinetics mediated exclusively by VEGF. In this study, we combined this Retro-tet system and LY333531, an inhibitor of the PKC-beta isoform, to elucidate the role of PKC-beta in tumor development and angiogenesis. Using a syngenic xenograft model, tumor augmentation induced by VEGF overexpression in HCC was markedly suppressed by oral administration of the PKC-beta inhibitor, with an accompanying reduction of neovascularization and p44/42 mitogen-activated protein kinase activation. This inhibitory effect was achieved even after the tumor was fully established. Immunohistochemical analysis revealed that apoptosis increased markedly in the tumor upon PKC-beta inhibitor treatment, whereas tumor cell proliferation itself did not change. Furthermore, with orthotopical transplantation, PKC-beta inhibition suppressed HCC tumor development in the liver. These results suggest that PKC-beta lies on the signal transduction pathway by which VEGF augments development and angiogenesis not only at the initial stage but also after the tumor is fully established.

The angiotensin-I-converting enzyme inhibitor perindopril suppresses tumor growth and angiogenesis: possible role of the vascular endothelial growth factor.

Angiotensin-I converting enzyme (ACE) inhibitor is used widely as an antihypertensive agent, and it has been suggested recently that it decreases the risk of cancer (A. F. Lever et al., Lancet, 352: 179-184, 1998). In this study, we examined the effect of several ACE inhibitors and angiotensin-II type 1 receptor (AT(1)-R) antagonists on tumor development and angiogenesis in a murine hepatocellular carcinoma model. Among ACE inhibitors, perindopril appeared to be a potent inhibitor of tumor development and angiogenesis, whereas AT(1)-R antagonists did not exert such an inhibitory effect. The inhibitory effect of perindopril was achieved even on established tumors. The level of the potent angiogenic factor, vascular endothelial growth factor (VEGF), in the tumor was significantly suppressed by perindopril. In vitro studies showed that perindopril-derived active form, perindoprilat, suppressed the endothelial cell tubule formation. Perindoprilat treatment also significantly inhibited VEGF mRNA expression in BNL-HCC cells in vitro. These results showed that the ACE inhibitor perindopril inhibited tumor development and angiogenesis independent from AT(1)-R blockage, and that VEGF alternation may be involved in the mechanism of this inhibitory effect. Because perindopril is widely used in clinical practice, it may represent an effective new strategy for anticancer therapy.

Construction of a Bacillus subtilis cloning vehicle with heterologous DNA sequence.

A cloning vehicle, pFTB91, for the Bacillus subtilis host was constructed with DNA fragments heterologous to the host chromosome. It consists of three DNA fragments: (i) chromosomal DNA of Bacillus amyloliquefaciens which complements the leuA and ilvC mutations in B. subtilis; (ii) a B. amyloliquefaciens plasmid DNA that supplies an autonomously replicating function; and (iii) a HindIII fragment of Staphylococcus aureus plasmid pTP5 that carries gene tetr, conferring the TetR phenotype. It has sufficiently low DNA homology to prevent its integration into the host chromosome in recombination-competent cells of B. subtilis. It is 9.3 kb, and approx. 10 copies are present per chromosome. The SalI and KpnI sites in the ilvC+ and tetr genes, respectively, could be used for selection of recombinant plasmids by insertional inactivation. The plasmid has unique sites for EcoRI, PstI, and XbaI.

Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions in the fibronectin finger-like domain and the kringle 1 domain.

Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent which is used to treat acute myocardial infarction. Pharmacokinetically, t-PA is characterized by a rapid clearance from the circulation. In a previous study, we constructed variant forms of t-PA with genetic modifications at the fibronectin finger-like domain (finger domain) or at the kringle 1 domain (K1 domain). The finger modified variant, t-PA N37S.S38V.G39V.R40E. A41F.Q42S had about a 6.0-fold higher plasma half-life in vivo than wild-type t-PA. Two variants with modifications in the K1 domain, t-PA G161R.K162R.S165W and t-PA N115P, showed an improved kinetic parameters and a 2.2-fold higher plasma half-life in vivo than wild-type t-PA, respectively. To create a recombinant variant of t-PA with a higher enzymatic activity and a further prolonged half-life in vivo, the genes containing each modifications were joined and expressed in animal cells. The two variants, t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G16 1R.K162R.S165W and t-PA N37S.S38V.G39V.R40E.A41F.Q42S.N11 5P, were purified from conditioned media and their biochemical, pharmacokinetic and thrombolytic profiles were investigated. Although the variant t-PA N37S.S38V.G39V.R40E.A41F.Q42S.G16 1R.K162R.S165W demonstrated an impaired enzymatic activity compared to the wild-type t-PA, the half-life of the variant, t-PA N37S.S38V.G39V.R40E.A41F.Q42S. N115P, following intravenous bolus injection in rabbits was considerably longer than that of finger-domain modified variants.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions in the finger domain.

Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7-9, 10-14, 15-19, 28-33, and 37-42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37-42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37-42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37-42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37-42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.