Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D H-1 NMR data, Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern), The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues, Both of these structural features may play a role in its binding to the voltage-gated sodium channel. (C) 1997 Federation of European Biochemical Societies.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications. (C) 2010 Elsevier GmbH. All rights reserved.

Protein crystallography and examples of its applications in medicinal chemistry

The field of protein crystallography inspires and enthrals, whether it be for the beauty and symmetry of a perfectly formed protein crystal, the unlocked secrets of a novel protein fold, or the precise atomic-level detail yielded from a protein-ligand complex. Since 1958, when the first protein structure was solved, there have been tremendous advances in all aspects of protein crystallography, from protein preparation and crystallisation through to diffraction data measurement and structure refinement. These advances have significantly reduced the time required to solve protein crystal structures, while at the same time substantially improving the quality and resolution of the resulting structures. Moreover, the technological developments have induced researchers to tackle ever more complex systems, including ribosomes and intact membrane-bound proteins, with a reasonable expectation of success. In this review, the steps involved in determining a protein crystal structure are described and the impact of recent methodological advances identified. Protein crystal structures have proved to be extraordinarily useful in medicinal chemistry research, particularly with respect to inhibitor design. The precise interaction between a drug and its receptor can be visualised at the molecular level using protein crystal structures, and this information then used to improve the complementarity and thus increase the potency and selectivity of an inhibitor. The use of protein crystal structures in receptor-based drug design is highlighted by (i) HIV protease, (ii) influenza virus neuraminidase and (iii) prostaglandin H-2-synthetase. These represent, respectively, examples of protein crystal structures that (i) influenced the design of drugs currently approved for use in the treatment of HIV infection, (ii) led to the design of compounds currently in clinical trials for the treatment of influenza infection and (iii) could enable the design of highly specific non-steroidal anti-inflammatory drugs that lack the common side-effects of this drug class.

Effects of the association zidovudine plus ritonavir on the liver and kidneys of pregnant rats. Morphological and biochemical aspects

Purpose: To evaluate biochemical and morphological effects on rats submitted to three different doses of the association zidovudine and ritonavir administered throughout pregnancy. Methods: Forty pregnant EPM-1 Wistar rats weighing about 200 g were randomly divided into the control group (Ctr = drug vehicle control, n = 10) and three experimental ones which were treated with an oral solution of zidovudine/ritonavir (Exp1 = 10/20 mg/kg bw, n = 10; Exp2 = 30/60 mg/kg bw, n = 10; Exp3 = 90/180 mg/kg bw, n = 10) from `day 0` up to the 20th day of pregnancy. At term (20th day) the rats were anesthetized. Blood and fetal and maternal organ samples (livers and kidneys) were taken for morphological and biochemical analyses. Results: Upon histological examinations fetal livers and kidneys appeared normal. In contrast the maternal samples revealed structural alterations. Maternal kidneys of the three experimental groups exhibited progressive and dose-dependent histological alterations; liver alterations were detected only in Exp3. Blood levels of AST and ALT were not significantly different from the control group but urea and creatinine levels were lower in groups Exp3 and Exp1. Conclusions: The administration of zidovudine plus ritonavir throughout rat pregnancy can cause morphological as well as functional changes in maternal kidneys.

A single nucleotide deletion at the C1 inhibitor gene as the cause of hereditary angioedema: insights from a Brazilian family

Background: Hereditary angioedema is an autosomal dominant disease characterized by episodes of subcutaneous and submucosal edema. It is caused by deficiency of the C1 inhibitor protein, leading to elevated levels of bradykinin. More than 200 mutations in C1 inhibitor gene have been reported. The aim of this study was to analyze clinical features of a large family with an index case of hereditary angioedema and to determine the disease-causing mutation in this family. Methods: Family pedigree was constructed with 275 individuals distributed in five generations. One hundred and sixty-five subjects were interviewed and investigated for mutation at the C1 inhibitor gene. Subjects reporting a history of recurrent episodes of angioedema and/or abdominal pain attacks underwent evaluation for hereditary angioedema. Results: We have identified a novel mutation at the C1 inhibitor gene, c.351delC, which is a single-nucleotide deletion of a cytosine on exon 3, resulting in frameshift with premature stop codon. Sequencing analysis of the hypothetical truncated C1 inhibitor protein allowed us to conclude that, if transcription occurs, this protein has no biological activity. Twenty-eight members of the family fulfilled diagnostic criteria for hereditary angioedema and all of them presented the c.351delC mutation. Variation in clinical presentation and severity of disease was observed among these patients. One hundred and thirty-seven subjects without hereditary angioedema did not have the c.351delC mutation. Conclusion: The present study provides definitive evidence to link a novel genetic mutation to the development of hereditary angioedema in patients from a Brazilian family.

Inhibition of Neutrophil Migration by Hemopexin Leads to Increased Mortality Due to Sepsis in Mice

Rationale: The reduction of neutrophil migration to the bacterial focus is associated with poor outcome in sepsis. Objectives: The objective of this study was to identify soluble substances in the blood of septic mice that inhibit neutrophil migration. Methods: A pool of serum obtained from mice 2 hours after the induction of severe sepsis by cecal ligation and puncture inhibited the neutrophil migration. The proteins with inhibitory activity on neutrophil migration were isolated by Blue-Sepharose chromatography, high-performance liquid chromatography, and electrophoresis, and identified by mass spectrometry. Measurements and Main Results: Hemopexin was identified as the serum component responsible for the inhibition of neutrophil migration. In sepsis, the pretreatment of wild-type mice with hemopexin inhibited neutrophil migration to the focus of infection and decreased the survival rate from 87.5 to 50.0%. Hemopexin-null mice subjected to severe sepsis presented normal neutrophil migration, low bacteremia, and an improvement of 40% in survival rate. Moreover, hemopexin inhibited the neutrophil chemotaxis response evoked by C5a or macrophage inflammatory protein-2 and induced a reduction of CXCR2 and L-selectin as well as the up-regulation of CD11b expression in neutrophil membranes. The inhibitory effect of hemopexin on neutrophil chemotaxis was prevented by serine protease inhibitors or ATP. In addition, serum levels of ATP were decreased 2 hours after severe sepsis. Conclusions: These data demonstrate for the first time the inhibitory role of hemopexin in neutrophil migration during sepsis and suggest that the therapeutic inhibition of hemopexin or its protease activity could improve neutrophil migration to the focus of infection and survival in sepsis.

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari : Ixodidae)

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation system. (c) 2008 Published by Elsevier Ltd.

State of the art etch-and-rinse adhesives

Objectives: The aim of this study was to explore the therapeutic opportunities of each step of 3-step etch-and-rinse adhesives. Methods: Etch-and-rinse adhesive systems are the oldest of the multi-generation evolution of resin bonding systems. In the 3-step version, they involve acid-etching, priming and application of a separate adhesive. Each step can accomplish multiple goals. Acid-etching, using 32-37% phosphoric acid (pH 0.1-0.4) not only simultaneously etches enamel and dentin, but the low pH kills many residual bacteria. Results: Some etchants include anti-microbial compounds such as benzalkonium chloride that also inhibits matrix metalloproteinases (MMPs) in dentin. Primers are usually water and HEMA-rich solutions that ensure complete expansion of the collagen fibril meshwork and wet the collagen with hydrophilic monomers. However, water alone can re-expand dried dentin and can also serve as a vehicle for protease inhibitors or protein cross-linking agents that may increase the durability of resin-dentin bonds. In the future, ethanol or other water-free solvents may serve as dehydrating primers that may also contain antibacterial quaternary ammonium methacrylates to inhibit dentin MMPs and increase the durability of resin-dentin bonds. The complete evaporation of solvents is nearly impossible. Significance: Manufacturers may need to optimize solvent concentrations. Solvent-free adhesives can seal resin-dentin interfaces with hydrophobic resins that may also contain fluoride and antimicrobial compounds. Etch-and-rinse adhesives produce higher resin-dentin bonds that are more durable than most 1 and 2-step adhesives. Incorporation of protease inhibitors in etchants and/or cross-linking agents in primers may increase the durability of resin-dentin bonds. The therapeutic potential of etch-and-rinse adhesives has yet to be fully exploited. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Solution structure of BSTI: A new trypsin inhibitor from skin secretions of Bombina bombina

The three-dimensional solution structure of BSTI, a trypsin inhibitor from the European frog Bombina bombina, has been solved using H-1 NMR spectroscopy. The 60 amino acid protein contains five disulfide bonds, which were unambiguously determined to be Cvs (4-38), Cys (13-34), Cys (17-30), Cys (21-60), and Cys (40-54) by experimental restraints and subsequent structure calculations. The main elements of secondary structure are four beta -strands, arranged as two small antiparallel beta -sheets, The overall fold of BSTI is disk shaped and is characterized by the lack of a hydrophobic core. The presumed active site is located on a loop comprising residues 21-34, which is a relatively disordered region similar to that seen in many other protease inhibitors. However, the overall fold is different to other known protease inhibitors with the exception of a small family of inhibitors isolated from nematodes of the family Ascaris and recently also from the haemolymph of Apis mellifera. BSTI may thus be classified as a new member of this recently discovered family of protease inhibitors.

A family of textilinin genes, two of which encode proteins with antihaemorrhagic properties

Two peptides, textilinins 1 and 2, isolated from the venom of the Australian common brown snake, Pseudonaja textilis textilis, are effective in preventing blood loss. To further investigate the potential of textilinins as anti-haemorrhagic agents, we cloned cDNAs encoding these proteins. The isolated full-length cDNA (430 bp in size) was shown to code for a 59 amino acid protein, corresponding in size to the native peptide, plus an additional 24 amino acid propeptide. Six such cDNAs were identified, differing in nucleotide sequence in the coding region but with an identical propeptide. All six sequences predicted peptides containing six conserved cysteines common to Kunitz-type serine protease inhibitors. When expressed as glutathione S-transferase (GST) fusion proteins and released by cleavage with thrombin, only those peptides corresponding to textilinin 1 and 2 were active in inhibiting plasmin with K-i values similar to those of their native counterparts and in binding to plasmin less tightly than aprotinin by two orders of magnitude. Similarly, in the mouse tail vein blood loss model only recombinant textilinin 1 and 2 were effective in reducing blood loss. These recombinant textilinins have potential as therapeutic agents for reducing blood loss in humans, obviating the need for reliance on aprotinin, a bovine product with possible risk of transmissible disease, and compromising the fibrinolytic system in a less irreversible manner.

Secretory pathway of trypanosomatid parasites

The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Tryanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles it? the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory), transport and protein glycosylation that may be exploited in developing new antiparasite drugs.

STANDARDIZATION OF PROCEDURES OF Plasmodium falciparum ANTIGEN PREPARATION FOR SEROLOGIC TESTS

The objective of the present study is to standardize the technical variables for preparation and storage of Plasmodium falciparum and of antigen components extracted with the amphoteric detergent Zwittergent. P. falciparum obtained from in vitro culture was stored at different temperatures and for different periods of time. For each variable, antigen components of the parasite were extracted in the presence or absence of protease inhibitors and submitted or not to later dialysis. Products were stored for 15, 30 and 60 days at different temperatures and immunological activity of each extract was determined by SDS-PAGE and ELISA using positive or negative standard sera for the presence of IgG directed to blood stage antigens of P. falciparum. Antigen extracts obtained from parasites stored at -20oC up to 10 days or at -70oC for 2 months presented the best results, showing well-defined bands on SDS-PAGE and Western blots and presenting absorbance values in ELISA that permitted safe differentiation between positive and negative sera.

Co-infection between influenza virus and flagellated bacteria

Trypsin is required in the hemagglutinin (HA) cleavage to in vitro influenza viruses activation. This HA cleavage is necessary for virus cell entry by receptor-mediated endocytosis. Bacteria in the respiratory tract are potential sources of proteases that could contribute to the cleavage of influenza virus in vivo. From 47 samples collected from horses, pigs, and from humans, influenza presence was confirmed in 13 and these samples demonstrated co-infection of influenza with flagellated bacteria, Stenotrophomonas maltophilia from the beginning of the experiments. Despite treatment with antibiotics, the bacteria remained resistant in several of the co-infected samples (48.39%). These bacteria, considered opportunistic invaders from environmental sources, are associated with viral infections in upper respiratory tract of hosts. The protease (elastase), secreted by Stenotrophomonas maltophilia plays a role in the potentiation of influenza virus infection. Proteolytic activity was detected by casein agar test. Positive samples from animals and humans had either a potentiated influenza infectivity or cytopathic effect (CPE) in MDCK and NCI H292 cells, Stenotrophomonas maltophilia were always present. Virus and bacteria were observed ultrastructurally. These in vitro findings show that microbial proteases could contribute to respiratory complications by host protease activity increasing inflammation or destroying endogenous cell protease inhibitors.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Prevalence of discordant immunologic and virologic responses in patients with AIDS under antiretroviral therapy in a specialized care center in Brazil

Some patients under antiretroviral therapy (ART) do not reach immune recovery when the viral load becomes undetectable. This is called discordant immunologic and virologic responses. Its prevalence varies between 8% and 24%. This study describes its prevalence and the characteristics of the affected subjects in the outpatient clinic of a Brazilian specialized-care center. Of 934 patients on ART, 536 had undetectable viral loads. Prevalence was 51/536 or 9% (95% confidence interval: 6.6% to 11.4%). Median age at the beginning of ART was 37 years (interquartile range - IQR: 31 to 45). Male gender and mixed race predominated (76.5% and 47.1% respectively). AIDS-defining illnesses were absent at the beginning of ART in 60.8%. Fifty-one percent were taking protease inhibitors, 43.2% Efavirenz and 5.8% both. Median time on ART was 36 months (IQR: 17-81 months). Irregular treatment was recorded for 21.6%. ART had been modified for 63% prior to the study, and 15.7% had used monotherapy or double therapy. Median CD4 count was 255 cells/mm³ (IQR: 200-284). Median viral load before ART was 4.7 log10 copies/mL (IQR: 4.5-5.2). Discordant responders were not different from AIDS patients in general, but there was a high frequency of multiple schedules of treatment.