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1.
Biol Reprod ; 109(6): 904-917, 2023 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-37712895

RESUMO

Cathepsin L plays physiological and pathological roles in immune responses, cancer, metamorphosis, and oogenesis in several species. However, the function of Cathepsin L in medaka ovaries remains unclear. Therefore, here, we examined the physiological functions of Cathepsin L in the medaka ovaries. Cathepsin L mRNA transcripts and proteins were found to be constitutively expressed in the ovaries of Oryzias latipes over a 24-h spawning cycle. Expression was localized within the oocyte cytoplasm of growing follicles and the follicle layer of preovulatory and postovulatory follicles. Moreover, the active form of Cathepsin L was highly expressed in the follicle layer of periovulatory follicles and the ovaries 2-6 h after ovulation. Recombinant Cathepsin L was activated under acidic conditions and exhibited enzymatic activity in acidic and neutral pH conditions. However, extracellular matrix proteins were degraded by recombinant Cathepsin L under acidic, not neutral pH conditions. Cathepsin L was secreted from preovulatory follicles, while active recombinant Cathepsin L was detected in the conditioned medium of a medaka cell line, OLHNI-2. Mechanistically, recombinant Cathepsin L activates recombinant urokinase-type plasminogen activator-1, which is expressed within the follicle layers post-ovulation. Meanwhile, the treatment of medakas with an E-64 or anti-Cathepsin L antibody effectively blocked follicular layer degeneration and degradation after ovulation, whereas in vitro ovulation was not inhibited by either. Collectively, the findings of this study indicate that although Cathepsin L does not impact ovulation in medakas, it contributes to the degeneration and degradation of the follicle layers following ovulation via activation of urokinase-type plasminogen activator-1, and not via the degradation of extracellular matrix proteins.


Assuntos
Oryzias , Ovário , Feminino , Animais , Ovário/fisiologia , Oryzias/fisiologia , Catepsina L/genética , Catepsina L/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/farmacologia , Ovulação/fisiologia , Proteínas da Matriz Extracelular
2.
Mol Biol Rep ; 50(4): 3935-3943, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36749527

RESUMO

BACKGROUND: Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis is one of the top thirteen causes of death worldwide. The major challenge to control TB is the emergence of drug-resistant tuberculosis (DR-TB); specifically, multi-drug resistant TB which are resistant to the most potent drugs; rifampin and isoniazid. Owing to the inconsistencies of the current diagnostic methods, a single test cannot identify the whole spectrum of DR-TB associated mutations. Recently, host blood transcriptomics has gained attention as a promising technique that develops disease-specific RNA signatures/biomarkers. However, studies on host transcriptomics infected with DR-TB is limited. Herein, we intended to identify genes/pathways that are differentially expressed in multi-drug/rifampin resistant TB (MDR/RR-TB) in contrast to drug susceptible TB. METHOD AND RESULTS: We conducted blood RNA sequencing of 10 pulmonary TB patients (4; drug susceptible and 6; DR-TB) and 55 genes that were differentially expressed in MDR/RR-TB from drug-susceptible/mono-resistant TB were identified. CD300LD, MYL9, VAMP5, CARD17, CLEC2B, GBP6, BATF2, ETV7, IFI27 and FCGR1CP were found to be upregulated in MDR/RR-TB in all comparisons, among which CLEC2B and CD300LD were not previously linked to TB. In comparison pathway analysis, interferon alpha/gamma response was upregulated while Wnt/beta catenin signaling, lysosome, microtubule nucleation and notch signaling were downregulated. CONCLUSION: Up/down-regulation of immunity related genes/pathways speculate the collective effect of hosts' attempt to fight against continuously multiplying DR-TB bacteria and the bacterial factors to fight against the host defense. The identified genes/pathways could act as MDR/RR-TB biomarkers, hence, further research on their clinical use should be encouraged.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Humanos , Rifampina/farmacologia , Projetos Piloto , Antituberculosos/farmacologia , Tuberculose/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/genética , Mycobacterium tuberculosis/genética , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana
3.
ACS Omega ; 7(50): 46494-46500, 2022 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-36570282

RESUMO

The development of antibacterial compounds using natural products, particularly nano-sized antibacterial products, has been intensively investigated in recent years. This study was conducted to compare the antibacterial activity of nanocurcumin with bulk curcumin against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria. Curcumin was extracted from turmeric rhizome using the Soxhlet extraction with ethanol. A physicochemical fabrication method was used to synthesize nanocurcumin from extracted curcumin. The particle size of nanocurcumin was 87 ± 8 nm. The 1H NMR spectrum of nanocurcumin show that all the peaks are well separated and can be interpreted to those of curcumin. According to the in vitro antibacterial assay, nanocurcumin shows better antibacterial activity against both Gram-positive and Gram-negative bacteria than bulk curcumin, with increased inhibition zones of 29.91 ± 0.53 mm (S. aureus) and 24.58 ± 1.12 mm (E. coli) when compared to 24.82 ± 0.54 mm (S. aureus) and 19.70 ± 1.18 mm (E. coli) of the latter. Subsequently, antibacterial creams were formulated, and the inhibition zones of nanocurcumin cream were larger than that of curcumin cream for both S. aureus and E. coli, exhibiting its superior antibacterial activity. Different storage periods of up to 1 month did not affect the inhibition zones significantly (p < 0.05), where nanocurcumin cream maintained its better antibacterial quality over bulk curcumin cream. There is no significant cytotoxicity in either of these formulations.

4.
Environ Res ; 203: 111779, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34339700

RESUMO

The epidemic of chronic kidney disease of unknown etiology (CKDu) that contributes significantly to morbidity and mortality rates among dry-zonal farming communities has become a public health priority in Sri Lanka. Though a large number of hypotheses were introduced as causative factors, none of them have been confirmed so far. As drinking water quality is among the most suspected causative factors for the emergence of CKDu, a detailed hydro-geochemical investigation was carried out concurrently with the population screening in the Monaragala district of Sri Lanka where high incidences of CKDu are reported. A population screening was performed selecting 46,754 people using both dipstick proteinuria test and Albumin-Creatinine Ratio (ACR). The results revealed that the disease prevalence is about 6.7 % in the district. A total of 60 groundwater samples, 30 each, were collected from CKDu-prevalent locations and control locations where there are no CKDu cases reported. The samples were analyzed to identify any possible linkage between water quality and disease prevalence. Concentrations of hardness, F-, Na+, and Mg2+ in groundwater revealed a statistically significant difference between CKDu and control wells at a confident level of p = 0.05. The study revealed that alkali (Na++K+) and alkaline earth cations (Mg2+, Ca2+, Sr2+, Ba2+) were relatively higher in drinking water sources used by CKDu patients, compared to the well waters used by healthy individuals. Nearly 87 % of the wells used by CKDu cases showed higher fluoride levels that exceed the threshold level (1.0 mg L-1). Contents of nephrotoxic trace elements such as As, Cd, and Pb were found to be comparable in both types of wells and were well below the WHO permissible levels, thus negating their prime influence on the CKDu prevalence. It is obtrusive that the elevated fluoride levels together with water hardness associated with higher Mg2+ levels have a possible relation with CKDu and may influence the disease progression.


Assuntos
Água Potável , Insuficiência Renal Crônica , Fluoretos/análise , Fluoretos/toxicidade , Dureza , Humanos , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/epidemiologia , Sri Lanka/epidemiologia
5.
Environ Geochem Health ; 42(2): 617-624, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31435865

RESUMO

Glutathione Peroxidase activity in whole blood is well correlated with the Selenium (Se) levels in cattle hence can be used effectively to assess the supply of Se to farm animals. In this study, Se status of cattle from five different geographic regions of Sri Lanka were assessed based on glutathione peroxidase (GSH-Px) activity. The GSH-Px activity was determined in whole blood samples collected from 80 cattle from 31 different farms in five districts viz. Kandy, Anuradhapura, Batticoloa, Trincomalee and Jaffna using photometric method. Mean GSH-Px activity was found to be 825, 1239, 1039, 849 and 1307 µkat L-1 in above districts, respectively while the reference value was considered as 665.4 µkat L-1. Among the studied animals, insufficient Se levels were detected in 50%, 17%, 9%, 27% and 5%, respectively, from above districts. Kruskal Wallis test indicated a significant variation among the sampled locations with respect to the GSH-Px activity (p = 0.001). Selenium content in pasture and water collected from studied locations varied from 6.0 to 554 µg kg-1 and < 0.03-1.14 µg L-1, respectively. The lower Se levels in feeds recorded from Kandy region infer the lower GSH-Px activity in the animals from the same region. This variability may be due to differences in nutrient supply, age and species of cattle, and lactation stage. Although the assessing method has some limitations, the activity of GSH-Px of the samples indirectly confirms that considerable numbers of cattle from Sri Lanka are with insufficient selenium levels.


Assuntos
Glutationa Peroxidase/sangue , Selênio/sangue , Ração Animal/análise , Animais , Disponibilidade Biológica , Bovinos , Feminino , Água Doce/análise , Água Subterrânea/análise , Masculino , Selênio/análise , Selênio/farmacocinética , Sri Lanka
6.
Springerplus ; 5(1): 1864, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27843741

RESUMO

BACKGROUND: Samples of 226 new improved and 21 indigenous rice (Oryza sativa L.) varieties were collected from the rice fields in three climatic zones of Sri Lanka and concentrations of 18 trace elements (Li, B, Al, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Ba, Pb and Bi) were measured giving particular emphasis on Se, Cd and As using ICP-MS. The two way multivariate analysis of variance (MANOVA) method was employed to identify the differences in composition among rice from different climatic zones. RESULTS: The mean values obtained for both white and red rice were Se (36; 25 µg/kg), As (42; 45 µg/kg) and Cd (70; 123 µg/kg) on dry weight basis. However mean content of Se, As and Cd of native rice varieties were 69, 74 and 33 µg/kg, respectively. Statistical interpretations showed that in the majority of cases, there was a significant difference in Cd content among climatic zones whereas Se and Pb show differences between white and red rice varieties. Arsenic did not indicate any significant difference either between rice types or among climatic regions. Notably Se and As contents in indigenous rice were higher than that of improved rice types. To assess the safety of dietary of intake, daily intake of Se, Cd and As by rice were calculated. Non-gender specific Estimated Daily Intake (EDI) of Se, Cd and As consuming improved rice are 9.31, 24.1 and 12.2 µg day-1, respectively. CONCLUSIONS: Since over 50 % of daily meals of people contain rice or rice based products, Se intake is expected to be deficient among the Sri Lankan population.

7.
Biol Reprod ; 92(1): 10, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25411388

RESUMO

We previously reported that the serine protease plasmin plays a role in follicle rupture during ovulation in the teleost medaka. In this study, we showed that urokinase-type plasminogen activator 1 (Plau1) is a physiological activator of plasminogen. Morphological analyses revealed that in the preovulatory follicle, plau1 mRNA was detected in association with follicle cells, while Plau1 protein was localized in the oocyte egg membrane. Both an inactive precursor and an active form of Plau1 were present at constant levels in the membrane fraction via the latter half of the 24-h spawning cycle. Plasminogen activator inhibitor-1 (Pai1) was detected in the follicle layer of the preovulatory follicle, but the protein level was low at approximately 7 h prior to ovulation. We showed that plasmin hydrolyzed laminin, which is a major component of the basement membrane and is situated between the granulosa and theca cells of the follicle. In vitro ovulation of large follicles was significantly inhibited by anti-Plau1 antibodies and active recombinant Pai1. Levels of Pai1 expression were increased in vivo at approximately 7 h prior to ovulation. Expression of Pai1 was also induced in vitro in the follicle with recombinant medaka luteinizing hormone (Lh). Lh-induced expression of pail mRNA was significantly suppressed by the presence of MDL (an adenylyl cyclase inhibitor), trilostane (a 3beta-hydroxysteroid dehydrogenase inhibitor), and RU486 (a nuclear progestin receptor antagonist). These results support our recent proposal of a sequential two-step ECM protein hydrolysis model for follicle rupture for medaka ovulation.


Assuntos
Oryzias/fisiologia , Folículo Ovariano/fisiologia , Ovulação/fisiologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Células HEK293 , Humanos , Hormônio Luteinizante/farmacologia , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Ovulação/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Ativador de Plasminogênio Tipo Uroquinase/genética
8.
Zoolog Sci ; 31(12): 840-8, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25483797

RESUMO

Previously, we reported that the medaka testis abundantly expresses the mRNA for trypsinogen, which is a well-known pancreatic proenzyme that is secreted into and activated in the intestine. Currently, we report our characterization of the medaka trypsin using a recombinant enzyme and show that this protein is a serine protease that shares properties with trypsins from other species. Two polypeptides (28- and 26-kDa) were detected in the testis extracts by Western blot analysis using antibodies that are specific for medaka trypsinogen. The 28-kDa polypeptide was shown to be trypsinogen (inactive precursor), and the 26-kDa polypeptide was shown to be trypsin (active protease). We did not detect enteropeptidase, which is the specific activator of trypsinogen, in the testis extract. Immunohistochemical analyses using the same trypsinogen-specific antibody produced a strong signal in the spermatogonia and spermatozoa of the mature medaka testis. Substantial staining was found with spermatocytes, whereas extremely weak signals were observed with spermatids. In vitro incubation of testis fragments with the trypsinogen antibody strongly inhibited the release of sperm from the testis into the medium. Trypsin activity was detected in sperm extracts using gelatin zymographic analysis. Immunocytochemistry showed that trypsinogen and trypsin were localized to the cell membranes surrounding the sperm head. Collectively, these results suggest that trypsin plays an important role in the testis function of the medaka.


Assuntos
Regulação da Expressão Gênica/fisiologia , Oryzias/metabolismo , Testículo/metabolismo , Tripsina/metabolismo , Tripsinogênio/metabolismo , Animais , Enteropeptidase/genética , Enteropeptidase/metabolismo , Imuno-Histoquímica , Masculino , Espermatozoides/fisiologia , Tripsina/genética , Tripsinogênio/genética
9.
PLoS One ; 8(1): e54482, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23372734

RESUMO

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare's serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.


Assuntos
Hormônio Luteinizante/genética , Oryzias/fisiologia , Folículo Ovariano/fisiologia , Ovulação/genética , RNA Mensageiro/genética , Receptores do LH/genética , Animais , Feminino , Regulação da Expressão Gênica , Gonadotropinas/farmacologia , Células HEK293 , Humanos , Hormônio Luteinizante/metabolismo , Metaloproteinase 15 da Matriz/genética , Metaloproteinase 15 da Matriz/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Oviparidade/fisiologia , Hipófise/fisiologia , Ligação Proteica , RNA Mensageiro/metabolismo , Receptores do LH/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas de Cultura de Tecidos
10.
Mol Cell Endocrinol ; 332(1-2): 67-77, 2011 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-20932877

RESUMO

In vitro ovulation of mature medaka ovarian follicles was inhibited by inhibitors of cyclooxygenase (COX) or by an antagonist of the prostaglandin E(2) receptor (EP). Of the three medaka COX genes, ptgs2 was most dominantly expressed in the fish ovary. The ptgs2 transcript was detected in all sizes of growing follicles. In a 24-h spawning cycle, large-sized follicles contained ptgs2 mRNA at a fairly constant level. The levels of COX enzyme activity and prostaglandin E(2) were also constant in the large-sized follicles during the spawning cycle. The expression of prostaglandin E(2) receptor EP4b (ptger4b) mRNA was drastically upregulated in the large-sized follicles as the ovulation time approached. The current results indicate that prostaglandin E(2), which might be produced by COX-2, is involved in the ovulation of medaka, and that EP4b is likely the receptor responsible for exerting the action of prostaglandin E(2) in the process.


Assuntos
Ciclo-Oxigenase 2/genética , Oryzias/anatomia & histologia , Oryzias/metabolismo , Ovário/metabolismo , Ovulação/fisiologia , RNA Mensageiro/metabolismo , Receptores de Prostaglandina E Subtipo EP4/genética , Sequência de Aminoácidos , Animais , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Feminino , Indometacina/metabolismo , Indometacina/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Oryzias/genética , Ovário/citologia , Ovulação/efeitos dos fármacos , RNA Mensageiro/genética , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Alinhamento de Sequência
11.
Mol Reprod Dev ; 74(8): 1053-63, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17219431

RESUMO

Tissue kallikrein mK1 is a serine protease involved in the generation of bioactive kinins for normal cardiac and arterial function in the mouse. In the present study, the tissue kallikrein gene Klk1, which codes for mK1, was shown to be one of the most prevalent of the Klk gene species in the uteri of adult mice, and its mRNA level was significantly higher at estrus than at diestrus. Klk1 mRNA expression was enhanced in the uteri of ovariectomized mice receiving estradiol-17beta treatment. Both endometrial epithelial and stromal cells isolated from the mice exhibited Klk1 expression at detectable levels when cultured in the presence of estradiol-17beta. mK1 was characterized using the recombinant active enzyme. mK1 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. Casein, gelatin, fibronectin, collagen type IV, and high-molecular-weight kininogen were degraded by mK1. The single-chain tissue-type plasminogen activator was converted to the two-chain form by mK1. In addition, mK1 degraded insulin-like growth factor binding protein-3. The present data suggest that mK1 may be implicated in the growth of uterine endometrial tissues during the proliferative phase.


Assuntos
Endométrio , Estrogênios/metabolismo , Calicreínas Teciduais/metabolismo , Útero/metabolismo , Animais , Células Cultivadas , Endométrio/anatomia & histologia , Endométrio/metabolismo , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Cininogênio de Alto Peso Molecular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Proteases/metabolismo , Calicreínas Teciduais/genética , Ativador de Plasminogênio Tecidual/metabolismo , Útero/citologia
12.
Zoolog Sci ; 24(8): 774-80, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18217483

RESUMO

Human kallikrein 14 (KLK14) is a member of the human kallikrein gene family of serine proteases, and its protein, hK14, has recently been suggested to serve as a new ovarian and breast cancer marker. To gain insights into hK14's physiological functions, the active recombinant enzyme was obtained in an enzymatically pure state for biochemical and enzymatic characterizations. We studied its substrate specificity and behavior to various protease inhibitors, and identified candidate physiological substrates. hK14 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type I, collagen type IV, fibrinogen, and high-molecular-weight kininogen. Furthermore, it rapidly hydrolyzed insulin-like growth factor binding protein-3 (IGFBP-3). These findings suggest that hK14 may be implicated in tumor progression in ovarian carcinoma.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Calicreínas/metabolismo , Neoplasias Ovarianas/metabolismo , Serina Endopeptidases/metabolismo , Biomarcadores Tumorais , Caseínas/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Feminino , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Gelatina/metabolismo , Humanos , Proteínas Recombinantes , Inibidores de Serina Proteinase , Especificidade por Substrato
13.
Zoolog Sci ; 23(11): 963-8, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17189908

RESUMO

Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (Klk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling.


Assuntos
Calicreínas/química , Calicreínas/metabolismo , Sequência de Aminoácidos , Animais , Escherichia coli , Expressão Gênica , Calicreínas/genética , Camundongos , Proteínas Recombinantes
14.
FEBS Lett ; 579(30): 6879-84, 2005 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-16337200

RESUMO

Human kallikrein 8 (KLK8) is a member of the human kallikrein gene family of serine proteases, and its protein, hK8, has recently been suggested to serve as a new ovarian cancer marker. To gain insights into the physiological role of hK8, the active recombinant enzyme was obtained in a pure state for biochemical and enzymatic characterizations. hK8 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type IV, fibrinogen, and high-molecular-weight kininogen. hK8 also converted human single-chain tissue-type plasminogen activator (65 kDa) to its two-chain form (32 and 33 kDa) by specifically cleaving the peptide bond Arg275-Ile276. This conversion resulted in a drastic increase in the activity of the activator toward the fluorogenic substrate Pyr-Gly-Arg-MCA and plasminogen in the absence of fibrin. Our findings suggest that hK8 may be implicated in ECM protein degradation in the area surrounding hK8-producing cells.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Calicreínas/química , Calicreínas/metabolismo , Arginina/química , Caseínas/química , Colágeno Tipo IV/química , Escherichia coli/genética , Feminino , Fibrinogênio/química , Fibronectinas/química , Gelatina/química , Humanos , Calicreínas/análise , Calicreínas/antagonistas & inibidores , Calicreínas/genética , Calicreínas/isolamento & purificação , Cinética , Proteínas Recombinantes de Fusão/metabolismo , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Ativador de Plasminogênio Tecidual/química , Ativador de Plasminogênio Tecidual/metabolismo
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