Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 µM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 µM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 µM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

A study of the interaction between Helicobacter pylori and components of the human fibrinolytic system

The interaction of plasminogen, tissue plasminogen activator (t-PA) and urokinase with a clinical strain of Helicobacter pylori was studied. Plasminogen bound to the surface of H. pylori cells in a concentration-dependent manner and could be activated to the enzymatic form, plasmin, by t-PA. Affinity chromatography assays revealed a plasminogen-binding protein of 58.9 kDa in water extracts of surface proteins. Surface-associated plasmin activity, detected with the chromogenic substrate CBS 00.65, was observed only when plasminogen and an exogenous activator were added to the cell suspension. The two physiologic plasminogen activators, t-PA and urokinase, were also shown to bind to and remain active on the surface of bacterial cells. epsilon-Aminocaproic acid caused partial inhibition of t-PA binding, suggesting that the kringle 2 structure of this activator is involved in the interaction with surface receptors. The activation of plasminogen by t-PA, but not urokinase, strongly depended on the presence of cells and a 25-fold enhancer effect on the initial velocity of activation by t-PA compared to urokinase was established. Furthermore, a relationship between cell concentration and the initial velocity of activation was demonstrated. These findings support the concept that plasminogen activation by t-PA on the bacterial surface is a surface-dependent reaction which offers catalytic advantages.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251™. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251™. Streptase™ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase™ and Solustrep™ formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251™ activity per vial, Streptase™ (75.7 ± 5.0 units) and Streptonase™ (94.7 ± 4.6 units) had the highest activity, while Unitinase™ (31.0 ± 2.4 units) and Strek™ (32.9 ± 3.3 units) had the weakest activity. Solustrep™ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

Uprootedness and the protection of migrants in the International Law of Human Rights

The article attempt to demonstrate the evolution of international law in connected to the subject of the forced immigrants'. The author supported by several texts, cases and resolutions of the regional level, through interamerican court and European court, and the global level, through the international court. It's shown the evolution that occurred in international law in millennium turn over, which recognize the immigrants' rights. However, it's stressed the necessity of the development of those laws connected to the theme e the recognition, from the States; the importance of law's that effort to ensure the respect to human rights relative to the immigrants and their families.

Human Rights promotion in Serbia: a difficult task for the European Union

This article explores the role of the European Union in the human rights protection, implementation and promotion in Serbia. It is clear that the EU demands on democratisation in the region of Western Balkans are crucial to achieve the respect for human rights. The human rights standards as part of the conditionality criteria of the EU is a clear message towards the countries aspiring membership. However, Serbia progress in the field has been difficult due to several internal constraints. This paper aims to uncover the democratisation process of Serbia on its path towards the EU, and its progress (or not) regarding human rights protection and implementation.

Economic sanctions and human rights: an analysis of competing enforcement strategies in Latin America

This article addresses the consequences of economic sanctions for the protection of human rights in Latin America. The literature on sanctions and compliance informs three hypotheses, which investigate the relationship between sanctions and the level of rights protection in two groups of countries: those that were targeted by sanctions and those that were not. Using data from the Political Terror Scale (PTS) and from Freedom House, I find empirical evidence that sanctions do improve the level of protection in countries that were not targeted. This finding can be explained by the deterrent effect attributed to sanctions by the compliance literature, broadly interpreted. The presence of economic sanctions in a given year increases the probability of observing better human rights practices by almost 50%. These results hold for the 12 Latin American countries that were not subject to economic sanctions for the period 1976-2004.

Human cryptosporidiosis: detection of specific antibodies in the serum by an indirect immunofluorescence

Cryptosporidium sp., a coccidian parasite usually found in the faeces of cattle, has been recently implicated as an agent of human intestinal disease, mainly in immunocompromised patients. In the study realized, by an indirect immunofluorescence technique, specific immunoglobulins (IgG and IgM) have been demonstrated in human serum against Cryptosporidium oocysts. Purified oocysts were used as antigens in the indirect immunofluorecence assay. After analyzing this test in sera from selected groups of patients, the frequency of both specific IgG and IgM of immunocompetent children who were excreting oocysts in their faeces was 62% and in children with negative excretion of oocysts was 20% and 40%, respectively. In adults infected with the human immunodeficiency virus (HIV) and who were excreting Cryptosporidium in their stools, the frequency was 57% for IgG but only 2% for IgM. Twenty three percent of immunocompromised adults with not determined excretion of oocysts in their stools had anti-Cryptosporidium IgG in their sera. Children infected with human immunodeficiency virus had no IgM and only 14% had IgG detectable in their sera. The indirect immunoflorescence assay, when used with other parasitological techniques appears to be useful for retrospective population studies and for diagnosis of acute infection. The humoral immune response of HIV positive patients to this protozoan agent needs clarification.

Human case of gastric infection by a fourth larval stage of Pseudoterranova decipiens (Nematoda, Anisakidae)

Only three cases of human infection by anisakid nematodes have been reported in Chile since 1976. In the present case, an anisakid worm, identified as a fourth-stage Pseudoterranova decipiens larva, was removed with a gastroendoscopic biopsy clipper from the stomach of a 45 year-old man from southern Chile. The patient, who presented acute epigastric pain and a continuous sensation of having an empty stomach, reported having eaten smoked fish. The worm was fixed in 70% ethanol and cleaned in lactophenol for morphological study. The morphometric characteristics of the worm are described and drawn. Anisakid larvae in fish flesh can be killed by freezing or cooking.

New cases of human diphyllobothriosis in Patagonia, Argentine

The purpose of the paper is to describe 4 new cases of human diphyllobothriosis in Patagonia, Argentine. Adult parasites recovered were submitted to morphological and histological analyses for taxonomic identification. The etiological agent found was always Diphyllobothrium latum and all the cases were autochthonous. These data combined with previous information make the number of autochthonous human cases of diphyllobothriosis registered in Argentina increase to 18.

Aedes albopictus may not be vector of dengue virus in human epidemics in Brazil

Over 60,500 dengue cases were reported in the state of Espírito Santo (ES), Brazil, between 1995 and 1998. The study's purpose was to identify whether Aedes albopictus was transmitting the dengue virus during an epidemic in the locality of Vila Bethânia (Viana County),Vitória, ES. From April 3 to 9, 1998, blood and serum samples were collected daily for virus isolation and serological testing. Four autochthonous cases were confirmed through DEN 1 virus isolation and two autochthonous cases through MAC ELISA testing. Of 37 Ae. aegypti and 200 Ae. albopictus adult mosquitoes collected and inoculated, DEN1 virus was isolated only from a pool of two Ae. aegypti female mosquitoes. The study results suggest that Ae. albopictus still cannot be considered an inter-human vector in dengue epidemics in Brazil.

Mental health research in Brazil: policies, infrastructure, financing and human resources

The objective of this descriptive study was to map mental health research in Brazil, providing an overview of infrastructure, financing and policies mental health research. As part of the Atlas-Research Project, a WHO initiative to map mental health research in selected low and middle-income countries, this study was carried out between 1998 and 2002. Data collection strategies included evaluation of governmental documents and sites and questionnaires sent to key professionals for providing information about the Brazilian mental health research infrastructure. In the year 2002, the total budget for Health Research was US$101 million, of which US$3.4 million (3.4) was available for Mental Health Research. The main funding sources for mental health research were found to be the São Paulo State Funding Agency (Fapesp, 53.2%) and the Ministry of Education (CAPES, 30.2%). The rate of doctors is 1.7 per 1,000 inhabitants, and the rate of psychiatrists is 2.7 per 100,000 inhabitants estimated 2000 census. In 2002, there were 53 postgraduate courses directed to mental health training in Brazil (43 in psychology, six in psychiatry, three in psychobiology and one in psychiatric nursing), with 1,775 students being trained in Brazil and 67 overseas. There were nine programs including psychiatry, neuropsychiatry, psychobiology and mental health, seven of them implemented in Southern states. During the five-year period, 186 students got a doctoral degree (37 per year) and 637 articles were published in Institute for Scientic Information (ISI)-indexed journals. The investment channeled towards postgraduate and human resource education programs, by means of grants and other forms of research support, has secured the country a modest but continuous insertion in the international knowledge production in the mental health area.