Crystal structure of human purine nucleoside phosphorylase at 2.3 angstrom resolution

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. In human, PNP is the only route for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and its low resolution structure has been used for drug design. Here we report the structure of human PNP solved to 2.3 Angstrom resolution using synchrotron radiation and cryocrystallographic techniques. This structure allowed a more precise analysis of the active site, generating a more reliable model for substrate binding. The higher resolution data allowed the identification of water molecules in the active site, which suggests binding partners for potential ligands. Furthermore, the present structure may be used in the new structure-based design of PNP inhibitors. (C) 2003 Published by Elsevier B.V.

Structure of human PNP complexed with ligands

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine-salvage pathway, which allows cells to utilize preformed bases and nucleosides in order to synthesize nucleotides. PNP is specific for purine nucleosides in the beta-configuration and exhibits a strong preference for purines containing a 6-keto group and ribosyl-containing nucleosides relative to the corresponding analogues. PNP was crystallized in complex with ligands and data collection was performed using synchrotron radiation. This work reports the structure of human PNP in complex with guanosine (at 2.80 angstrom resolution), 3' deoxyguanosine (at 2.86 angstrom resolution) and 8-azaguanine (at 2.85 angstrom resolution). These structures were compared with the PNP-guanine, PNP-inosine and PNP-immucillin-H complexes solved previously.

Determining the structural basis for specificity of ligands using crystallographic screening

Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.

Structural basis for inhibition of human PNP by immucillin-H

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation. This work reports on the crystallographic study of the complex of human PNP-immucillin-H (HsPNP-ImmH) solved at 2.6 Angstrom resolution using synchrotron radiation. Immucillin-H (ImmH) inhibits the growth of malignant T-cell lines in the presence of deoxyguanosine without affecting non-T-cell tumor lines. ImmH inhibits activated normal human T cells after antigenic stimulation in vitro. These biological effects of ImmH suggest that this agent may have utility in the treatment of certain human diseases characterized by abnormal T-cell growth or activation. This is the first structural report of human PNP complexed with immucillin-H. The comparison of the complex HsPNP-ImmH with recent crystallographic structures of human PNP explains the high specificity of immucillin-H for human PNP. (C) 2003 Elsevier B.V. All rights reserved.

Contamination by chlorinated pesticides, PCBs and PBDEs in Atlantic spotted dolphin (Stenella frontalis) in western South Atlantic

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Anatomy, ultrastructure and secretion of Hibiscus pernambucensis Arruda (Malvaceae) extrafloral nectary

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito da utilização de diferentes diluidores para a produção in vitro de embriões bovinos

Objetivou-se avaliar as características morfológica e funcional do sêmen bovino congelado comparando-se a eficácia de dois diferentes diluidores. O ejaculado de quatro touros foi dividido em duas partes iguais, uma submetida ao diluidor Tris e gema de ovo (A) e outra ao diluidor à base de lecitina de soja (Andromed®) (B). No experimento I, cinco palhetas dos diluidores A e B de cada touro foram descongeladas e avaliadas quanto à motilidade, vigor, concentração, morfologia espermática e teste de termor-resistência lento. Foram feitas, ainda, avaliação da integridade de membranas, por meio da associação das sondas iodeto de propídio, isotiocionato de fluoresceína - Pisum sativum e carbocianina catiônica lipofílica, e avaliação funcional da membrana plasmática com teste hiposmótico. A avaliação da integridade da cromatina foi realizada pelo método de coloração com laranja de acridina. No experimento II, o sêmen com os diferentes diluidores foi utilizado na fecundação in vitro, sendo observadas taxas de clivagem e desenvolvimento embrionário in vitro. em relação aos resultados obtidos, apenas a porcentagem de espermatozoides no sêmen congelado foi discretamente maior com o diluidor A, concluindo-se que o diluidor composto por lecitina de soja pode substituir o composto por Tris e gema de ovo, respeitando-se as variações individuais de cada touro utilizado no presente experimento.

Fractionation and identification of the nucleic acid-related substances secreted by Streptomyces aureofaciens

S. aureofaciens growth in a chemically defined medium was associated with the active secretion of nucleic acid-related substances in the medium. High secretion depended on low availability of phosphate, and fractionation showed 7 anionic substances were secreted as major components. When compared to 76 known purine and pyrimidine derivatives only erotic acid was identified. Cationic components are among the minor concentration components secreted which have been identified as cytosine, inosine, cytidine, adenine, guanine and, probably, 1-methyl-adenine.

Effect of epicuticular waxes of fruits on the photodegradation of fenthion

After treatment lipophilic pesticides tend to diffuse by penetrating the epicuticular wax of fruits. In this way, solar radiation only acts on pesticide molecules after passing through the waxes. The effect of epicuticular waxes of three fruits (orange, nectarine, and olive) on the photodegradation of fenthion was studied. The waxes affected the photodegradation process of fenthion. The decay rate of fenthion increased in the presence of orange and nectarine waxes, while it decreased when olive wax was used. In all waxes, the transformation of fenthion produced mainly fenthion sulfoxide and low amounts of fenthion sulfone. In orange wax, 50% of the initial fenthion was transformed into unknown compounds. In nectarine wax, fenthion was degraded stoichiometrically into fenthion sulfoxide and fenthion sulfone. In olive wax, the photodegradation of fenthion yielded about 80% of fenthion sulfoxide.

Purine nucleoside phosphorylase: A potential target for the development of drugs to treat T-cell- and apicomplexan parasite-mediated diseases

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and thus drugs that inhibit human PNP activity have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Besides, the purine salvage pathway is the only possible way for apicomplexan parasites to obtain the building blocks for RNA and DNA synthesis, which makes PNP from these parasites an attractive target for drug development against diseases such as malaria. Hence, a number of research groups have made efforts to elucidate the mechanism of action of PNP based on structural and kinetic studies. It is conceivable that the mechanism may be different for PNPs from diverse sources, and influenced by the oligomeric state of the enzyme in solution. Furthermore, distinct transition state structures can make possible the rational design of specific inhibitors for human and apicomplexan enzymes. Here, we review the current status of these research efforts to elucidate the mechanism of PNP-catalyzed chemical reaction, focusing on the mammalian and Plamodium falciparum enzymes, targets for drug development against, respectively, T-Cell and Apicomplexan parasites-mediated diseases.

The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the air-water interface

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Associacao de opioides lipofilicos a bupivacaina na anestesia peridural. Ha vantagem no aumento da dose do opioide?

Background and Objectives: - The effects of associating lipophilic opioids to local anesthetics in epidural anesthesia are not well defined. There are still questions and controversies about opioid doses to be used and their major effects in the epidural block. This study aimed at evaluating the epidural block effects in humans of the association of different fentanyl and sufentanil doses to bupivacaine with 1:200.000 epinephrine. Methods: - A double-blind randomized study was performed in 94 patients of both genders, physical status ASA I, aged between 18 and 60 years, submitted to lower abdomen, perineal or lower limb surgery. Patients without preanesthetic medication were epidurally injected with 100 mg (20 ml) 0.5% bupivacaine, 0.1 mg (0.1 ml) 1%o epinephrine plus a combination of the following drugs: BUPI Group (15 patients): 2 ml of 0.9% saline solution (SS); FENT50 Group (19 patients): 50 μg (1 ml) fentanyl + 1 ml SS; FENT100 Group (20 patients): 100 μg (2 ml) fentanyl; SUF30 Group (20 patients): 30 μg (0.6 ml) sufentanil + SS (1.4 ml); SUF100 Group (20 patients): 50 μg (1 ml) sufentanil + SS (1 ml). The following parameters were studied: onset of sensory block, analgesic block (onset time) in T12, T10 and T8, analgesic block duration in T10 and T12, motor block degree, consciousness degree, need for supplemental perioperative sedation and analgesia, hypotension, bradycardia and peri and post operative side-effects, analgesia duration, proportion of patients needing supplemental analgesia and evaluation of postoperative pain (pain analog visual scale). Results: Groups were demographically uniform. The addition of fentanyl or sufentanil did not alter major characteristics of perioperative epidural block and has not significantly increased postoperative analgesia duration as compared to the use of bupivacaine only. However, the addition of lipophilic opioids has increased the quality of perioperative anesthetic block, translated into a lesser need for supplemental analgesia (p < 0.02). The increased dose of fentanyl and especially of sufentanil has increased the incidence of perioperative drowsiness (p < 0.001) without significant increase in other side effects. Conclusions: In the conditions and doses used, the addition of lipophilic opioids to bupivacaine and the increased dose of lipophilic opioids have improved anesthetic block quality without changes in the epidural block characteristics or a significant increase in side effects, with the exception of drowsiness mainly caused by sufentanil. However, they were not able to provide a significant increase in postoperative analgesia duration.

Preparation of N2, N2,7-trimethylguanosine affinity columns

2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns. TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs. The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG- binding proteins were obtained. The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs.

Encapsulação da rifampicina em matrizes poliméricas flutuantes obtidas por liofilização a partir de metilcelulose e ftalato de hidroxipropilmetilcelulose. Avaliação da liberação in vitro

Floating multiparticles for oral administration with different compositions were studied from a matricial polymeric system to obtain sustained release. The polymers used in the multiparticles constitution were methylceullose (MC) and hydroxypropylmethylcelullose phthalate (HPMCP) in several proportions. Spherical and isolated structures were obtained using HPMCP/MC in the range from 1:3 to 1: 13. The diameters of the floating multiparticles were in the range from 3 to 3.25 mm, while the non-floating particles were between 1.75 and 2.1 mm. The morphological analysis by confocal microscopy showed that the probable mechanism of drug release was the diffusion from the inner of particles to external media. The encapsulation of hydrophilic model substances (tartrazin and bordeaux S), showed that the maximum incorporation was about 38%, while for the lipophilic model substances (rifampicin) was 45%. The in vitro release of rifampicin in acid medium was dependent on the ratio HPMCP/MC. In alkaline medium the release followed a two-step profile, with slow release in the initial times and subsequent increase in the higher times The initial drug delivery profile was not dependent on the MC/HPMCP ratio and can be related with the release of the antibiotic from multiparticle inner caused by the swelling of polymers by the presence of water in the system. However, afterwards the release proceeds with typical profile of process involving hydrogels systems.

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg -1. © 2002 Elsevier Science (USA). All rights reserved.