Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms.

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1-130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1. SIGNIFICANCE: The comparative analysis established an array of specific proteins in pathogenic strain that will narrow down the identification of immune protective proteins that will help fight leptospirosis.

Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1–130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1.

Unveiling Lung Adenocarcinoma: Non-bacterial Thrombotic Endocarditis as the Debut Sign.

Non-bacterial thrombotic endocarditis (NBTE) involves the deposition of fibrin and platelets on heart valves, frequently leading to systemic embolism. The association between NBTE and cancer demands thorough investigation in cases lacking an evident cause. This case report elucidates the clinical course of a nonsmoking woman in her sixties with NBTE linked to pulmonary adenocarcinoma. The patient, who had a history of multiple sclerosis (MS) and was receiving dimethyl fumarate treatment, presented to the emergency department with stroke-like symptoms. Diagnostic challenges arose due to preexisting motor sensory impairment from MS. Initial evaluations revealed hypocapnia and elevated inflammatory markers. Blood cultures were obtained twice, and imaging confirmed pneumonia, left pleural effusion, and chronic pulmonary embolism while excluding acute vascular events or intracranial hemorrhage. The first transthoracic echocardiogram (TTE) indicated no cardiac abnormalities. Treatment encompassed parenteral antibiotics, systemic anticoagulation, and admission to medical floors. Although the initial treatment yielded a positive clinical response, subsequent complications emerged. On the tenth day, the patient required additional interventions, including broad-spectrum antibiotics and supplemental oxygen. A follow-up chest X-ray revealed persistent pneumonia and pleural effusion, and blood cultures upon admission returned negative. A subsequent head MRI confirmed an embolic stroke and displayed evidence of MS progression. Around the twentieth day, empirical treatment for infective endocarditis was initiated, and an 8 mm vegetation on the aortic valve was identified via transesophageal echocardiography (TOE). Acute pulmonary edema prompted a transfer to the intermediate care unit. Further investigations, including left thoracocentesis and CT, unveiled exudate and metastatic lesions in the liver, ilium, and kidney. Unfortunately, on the twenty-fifth day, the patient experienced acute myocardial infarction, right leg ischemia, disseminated intravascular coagulation, and shock. Pleural fluid analysis revealed malignant cells suggestive of lung adenocarcinoma. This case underscores the pivotal role of timely NBTE recognition and the search for malignancy when workup for infective endocarditis and autoimmune panels is negative. Moreover, it emphasizes the significance of vigilant monitoring, particularly in immunocompromised individuals or those with preexisting neurological deficits, especially when new neurological symptoms manifest. These insights significantly contribute to the comprehension of NBTE management and its implications for analogous patient cohorts.

Antimicrobial Activity of Peptides Produced by Lactococcus lactis subsp. lactis on Swine Pathogens.

Swine production is of great importance worldwide and has huge economic and commercial impact. Due to problems with bacterial infection, the use of antimicrobials has increased in the last decades, particularly in Latin America and Asia. This has led to concerns about antimicrobial resistance, which poses risks to human health and the environment. The use of probiotic organisms has been proposed as an alternative to this use, as these beneficial bacteria can produce antimicrobial peptides, such as bacteriocins, which allow the induction of inhibitory effects against pathogenic microorganisms. Among probiotics, some bacteria stand out with the inhibition of animal pathogens. The bacteriocin-like inhibitory substances (BLISs) of Lactococcus lactis subsp. lactis strain L2, present in its cell-free supernatant, were tested against pathogenic strains isolated from pig samples, such as Escherichia coli, Salmonella enterica, Streptococcus suis, Streptococcus dysgalactiae, Staphylococcus hyicus, and Enterococcus faecalis. Compounds secreted by L. lactis L2 have been shown to inhibit the growth of some pathogenic species, particularly Gram-positive bacteria, with S. suis being the most prominent. Antimicrobial peptides with a molecular size of 500-1160 Daltons were isolated from BLISs. The results highlight the potential of L. lactis BLISs and its peptides as natural antimicrobials for use in the food industry and to reduce the use of growth promoters in animal production.

Functional Status Following Pulmonary Rehabilitation: Responders and Non-Responders.

The 6 min walking test (6MWT) has been largely studied. Less is, however, known about responders and non-responders to pulmonary rehabilitation (PR) in other meaningful activities. We explored responders and non-responders and the predictors of response to PR in the 1 min sit-to-stand test (1 min STS) and the 6MWT and compared both measures in classifying responders. An observational study was conducted with 121 people with chronic obstructive pulmonary disease (COPD). The functional status was assessed before and after PR. Baseline differences between responders and non-responders were tested with Mann-Whitney U, chi-square, or Fisher exact tests. Predictors were explored with binary logistic regressions. Agreement between both measures was assessed with chi-square, Cohen's kappa, and McNemar tests. There were 54.5% and 57.0% of responders in the 1 min STS and the 6MWT, respectively. The proportion of responders was significantly different (p = 0.048), with a small agreement between the measures (kappa = 0.180; p = 0.048). The baseline 6MWT was the only significant predictor of response in the 6MWT (OR = 0.995; pseudo-r2 = 0.117; p < 0.001). No significant predictors were found for the 1 min STS. A large number of non-responders in terms of functional status exist. The 1 min STS and the 6MWT should not be used interchangeably. Future studies should explore the added benefit of personalizing PR to this outcome and investigate other potential predictors.

Instituições de longa permanência como alternativa no acolhimento das pessoas idosas / Long stay institutions as an alternative for protecting the elderly / Entidades de larga permanencia como alternativa para acoger adultos mayores

RESUMO Estudo reflexivo que enfatiza a Instituição de Longa Permanência para Idosos. Considerando a carga contextual em que se dá o processo de institucionalização em uma Instituição de Longa Permanência para Idosos, o qual é escoltado por transformações na vivência da pessoa idosa. Nessa perspectiva este estudo tem como propósito refletir sobre as Instituições de Longa Permanência como alternativa no acolhimento das pessoas idosas brasileiras. Primeiramente, expõe-se sobre o envelhecimento populacional, seguido por uma breve perspectiva histórica das Instituições de Longa Permanência e o finaliza com o processo de institucionalização da pessoa idosa nas mesmas. Embora existam definições a respeito das funções e obrigações da Instituição de Longa Permanência, a influência dos aspectos negativos na vivência das pessoas idosas institucionalizadas encontra-se implícita na maioria delas, solicitando uma reestruturação desse ambiente que tem se tornado o mundo-vida de uma demanda crescente de pessoas idosas. O processo de institucionalização e a vivência do idoso na instituição são elementos de estudo fundamentais para o alcance de um entendimento aprofundado da realidade intrínseca dessa pessoa que está escrevendo o epílogo da sua história de vida.(AU)

ABSTRACT This is a reflective study on long stay institutions for elderly population. Considering the context in which the process of institutionalization in a long-stay institution for the elderly occurs, which is escorted by changes in every-day life of the elderly population, this study aims to reflect on the long-stay institutions as an alternative of sheltering for the Brazilian elderly. First, demographic aging is analyzed, followed by a brief historical account regarding long-stay institutions, and an approach to the institutionalization process of the elderly population in these places. Although the functions and duties of long-stay institutions have been previously defined, the negative aspects that influence the experience of institutionalized elderly are evident; therefore, restructuring the environment of these type of places is necessary considering the growing demand. The institutionalization process and the experience of the elderly population admitted to these institutions are key study elements to comprehend the intrinsic reality of this population.(AU)

RESUMEN El presente es un estudio reflexivo sobre las instituciones de larga permanencia para adultos mayores. Con base en la carga contextual que se genera alrededor del proceso de institucionalización de estas personas, el cual implica una serie de cambios en su vida cotidiana, este estudio tiene como objetivo hacer una reflexión sobre la pertinencia de estos lugares como alternativa para acoger adultos mayores brasileños. En primer lugar, se hace un análisis sobre el proceso de envejecimiento de la población, seguido de un breve recuento histórico de las instituciones de larga permanencia, para finalizar con una descripción sobre el proceso de institucionalización. Si bien hay definiciones acerca de las funciones y obligaciones de este tipo de instituciones, los aspectos negativos que influyen en las experiencias de las personas mayores ya institucionalizadas son evidentes. En este sentido, es necesario reestructurar ese ambiente que se convierte en el diario vivir de estas personas, de el cual tiene una demanda cada vez mayor. El proceso de institucionalización y las experiencias de las personas mayores dentro la institución son elementos de estudio fundamentales para comprender a profundidad la realidad intrínseca de esta población.(AU)

Bioleaching of electronic waste using bacteria isolated from the marine sponge Hymeniacidon heliophila (Porifera).

The bacteria isolated from Hymeniacidon heliophila sponge cells showed bioleaching activity. The most active strain, Hyhel-1, identified as Bacillus sp., was selected for bioleaching tests under two different temperatures, 30°C and 40°C, showing rod-shaped cells and filamentous growth, respectively. At 30°C, the bacteria secreted substances which linked to the leached copper, and at 40°C metallic nanoparticles were produced inside the cells. In addition, infrared analysis detected COOH groups and linear peptides in the tested bacteria at both temperatures. The Hyhel-1 strain in presence of electronic waste (e-waste) induced the formation of crust, which could be observed due to bacteria growing on the e-waste fragment. SEM-EDS measurements showed that the bacterial net surface was composed mostly of iron (16.1% w/w), while a higher concentration of copper was observed in the supernatant (1.7% w/w) and in the precipitated (49.8% w/w). The substances linked to copper in the supernatant were sequenced by MALDI-TOF-ms/ms and identified as macrocyclic surfactin-like peptides, similar to the basic sequence of Iturin, a lipopeptide from Bacillus subtilis. Finally, the results showed that Hyhel-1 is a bioleaching bacteria and cooper nanoparticles producer and that this bacteria could be used as a copper recovery tool from electronic waste.

[Long stay institutions as an alternative for protecting the elderly]. / Instituições de longa permanência como alternativa no acolhimento das pessoas idosas.

This is a reflective study on long stay institutions for elderly population. Considering the context in which the process of institutionalization in a long-stay institution for the elderly occurs, which is escorted by changes in every-day life of the elderly population, this study aims to reflect on the long-stay institutions as an alternative of sheltering for the Brazilian elderly. First, demographic aging is analyzed, followed by a brief historical account regarding long-stay institutions, and an approach to the institutionalization process of the elderly population in these places. Although the functions and duties of long-stay institutions have been previously defined, the negative aspects that influence the experience of institutionalized elderly are evident; therefore, restructuring the environment of these type of places is necessary considering the growing demand. The institutionalization process and the experience of the elderly population admitted to these institutions are key study elements to comprehend the intrinsic reality of this population.

Estudo reflexivo que enfatiza a Instituição de Longa Permanência para Idosos. Considerando a carga contextual em que se dá o processo de institucionalização em uma Instituição de Longa Permanência para Idosos, o qual é escoltado por transformações na vivência da pessoa idosa. Nessa perspectiva este estudo tem como propósito refletir sobre as Instituições de Longa Permanência como alternativa no acolhimento das pessoas idosas brasileiras. Primeiramente, expõe-se sobre o envelhecimento populacional, seguido por uma breve perspectiva histórica das Instituições de Longa Permanência e o finaliza com o processo de institucionalização da pessoa idosa nas mesmas. Embora existam definições a respeito das funções e obrigações da Instituição de Longa Permanência, a influência dos aspectos negativos na vivência das pessoas idosas institucionalizadas encontra-se implícita na maioria delas, solicitando uma reestruturação desse ambiente que tem se tornado o mundo-vida de uma demanda crescente de pessoas idosas. O processo de institucionalização e a vivência do idoso na instituição são elementos de estudo fundamentais para o alcance de um entendimento aprofundado da realidade intrínseca dessa pessoa que está escrevendo o epílogo da sua história de vida.

El presente es un estudio reflexivo sobre las instituciones de larga permanencia para adultos mayores. Con base en la carga contextual que se genera alrededor del proceso de institucionalización de estas personas, el cual implica una serie de cambios en su vida cotidiana, este estudio tiene como objetivo hacer una reflexión sobre la pertinencia de estos lugares como alternativa para acoger adultos mayores brasileños. En primer lugar, se hace un análisis sobre el proceso de envejecimiento de la población, seguido de un breve recuento histórico de las instituciones de larga permanencia, para finalizar con una descripción sobre el proceso de institucionalización. Si bien hay definiciones acerca de las funciones y obligaciones de este tipo de instituciones, los aspectos negativos que influyen en las experiencias de las personas mayores ya institucionalizadas son evidentes. En este sentido, es necesario reestructurar ese ambiente que se convierte en el diario vivir de estas personas, de el cual tiene una demanda cada vez mayor. El proceso de institucionalización y las experiencias de las personas mayores dentro la institución son elementos de estudio fundamentales para comprender a profundidad la realidad intrínseca de esta población.

Clinical role of the nurse: concept analysis.

AIMS AND OBJECTIVES: To identify the attributes of the concept 'clinical role of the nurse' in the literature. BACKGROUND: The concept of nurses' clinical role is frequently mentioned in literature, but hardly explored in conceptual terms. This has implications for nursing practice and education. DESIGN: Qualitative and descriptive study, which corresponds to the first phase in the concept development of the qualitative concept analysis method - identification of concept attributes. METHODS: The critical literature analysis method was used to identify the antecedents, defining attributes and consequences of the nurse's clinical role. A systematic literature search was undertaken in International Literature in Health Sciences, Cumulative Index to Nursing and Allied Health Literature and Latin American and Caribbean Health Sciences. RESULTS: The clinical role was shown to be a process of complex interaction between nurse and patient, with critical thinking, informed experience and a sense of clinical autonomy as its antecedents. Consequences of nurses' clinical role include transformations in the organisation and process of nursing practice. A theoretical proposal was elaborated for the concept of the clinical role of the nurse, identifying the defining attributes, antecedents and consequences. CONCLUSIONS: The clinical role of the nurse concept that was developed represents innovative evidence on the theme. Nevertheless, a deeper understanding of nurses' clinical role is needed, as well as refinement of its conceptual components. This study should be integrated into a field research project, designed to illuminate how nurses manifest and articulate the concept in clinical practice. RELEVANCE TO CLINICAL PRACTICE: Knowledge of clinical role attributes, associated with nursing competencies, can contribute to reflection on the dimensions involved in nursing practice and inform not only teaching and professional practice, but also health policies.

The venomous secrets of the web droplets from the viscid spiral of the orb-weaver spider Nephila clavipes (Araneae, Tetragnatidae).

The capture web of N. clavipes presents viscous droplets, which play important roles in web mechanics and prey capture. By using scanning and transmission electron microscopy, it was demonstrated that the web droplets are constituted of different chemical environments, provided by the existence both of an aqueous and a lipid layer, which, in turn, present a suspension of tenths of vesicles containing polypeptides and/or lipids. GC/EI-MS Analysis of the contents of these vesicles led to the identification of some saturated fatty acids, such as decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, octadecanoic acid, and icosanoic acid, while other components were unsaturated fatty acids, such as (Z)-tetradec-9-enoic acid, (Z)-octadec-9-enoic acid, and (Z)-icosa-11-enoic acid; and polyunsaturated fatty acids like (9Z,12Z)-octadeca-9,12-dienoic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid, and (11Z,14Z)-icosa-11,14-dienoic acid. Toxic proteins such as calcium-activated proteinase and metalloproteinase jararhagin-like precursor were also identified by using a proteomic approach, indicating the possible involvement of these enzymes in the pre-digestion of spiders' preys web-captured. Apparently, the mixture of fatty acids are relatively toxic to insects by topical application (LD50 64.3+/-7.6 ng mg(-1) honeybee), while the proteins alone present no topical effect; however, when injected into the prey-insects, these proteins presented a moderate toxicity (LD50 40.3+/-4.8 ng mg(-1) honeybee); the mixture of fatty acids and proteins is very toxic to the preys captured by the web droplets of the viscid spiral of Nephila clavipes when topically applied on them (LD50 14.3+/-1.8 ng mg(-1) honeybee).

Functional shikimate dehydrogenase from Mycobacterium tuberculosis H37Rv: purification and characterization.

Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded shikimate dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis shikimate dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents.

Isolation and chemical characterization of PwTx-II: a novel alkaloid toxin from the venom of the spider Parawixia bistriata (Araneidae, Araneae).

Brazil has many species of spiders belonging to Araneidae family however, very little is known about the composition, chemical structure and mechanisms of action of the main venom components of these spiders. The main objective of this work was to isolate and to perform the chemical characterization of a novel beta-carboline toxin from the venom of the spider Parawixia bistriata, a typical species of the Brazilian 'cerrado'. The toxin was purified by RP-HPLC and structurally elucidated by using a combination of different spectroscopic techniques (UV, ESI-MS/MS and 1H NMR), which permitted the assignment of the molecular structure of a novel spider venom toxin, identified as 1-4-guanidinobutoxy-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline, and referred to here as PwTx-II. This compound is toxic to insects (LD50 = 12+/-3 etag/mg honeybee), neurotoxic, convulsive and lethal to rats (LD50 = 9.75 mg/kg of male Wistar rat).

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista.

Two novel inflammatory peptides were isolated from the venom of the social wasp Polybia paulista. They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as: Polybia-MPI: I D W K K L L D A A K Q I L-NH2 (1654.09 Da), Polybia-CP: I L G T I L G L L K S L-NH2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations.

DAHP synthase from Mycobacterium tuberculosis H37Rv: cloning, expression, and purification of functional enzyme.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains the leading cause of mortality due to a bacterial pathogen. According to the 2004 Global TB Control Report of the World Health Organization, there are 300,000 new cases per year of multi-drug resistant strains (MDR-TB), defined as resistant to isoniazid and rifampicin, and 79% of MDR-TB cases are now "super strains," resistant to at least three of the four main drugs used to treat TB. Thus there is a need for the development of effective new agents to treat TB. The shikimate pathway is an attractive target for the development of antimycobacterial agents because it has been shown to be essential for the viability of M. tuberculosis, but absent from mammals. The M. tuberculosis aroG-encoded 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (mtDAHPS) catalyzes the first committed step in this pathway. Here we describe the PCR amplification, cloning, and sequencing of aroG structural gene from M. tuberculosis H37Rv. The expression of recombinant mtDAHPS protein in the soluble form was obtained in Escherichia coli Rosetta-gami (DE3) host cells without IPTG induction. An approximately threefold purification protocol yielded homogeneous enzyme with a specific activity value of 0.47U mg(-1) under the experimental conditions used. Gel filtration chromatography results demonstrate that recombinant mtDAHPS is a pentamer in solution. The availability of homogeneous mtDAHPS will allow structural and kinetics studies to be performed aiming at antitubercular agents development.

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis.

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aroA-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1L of LB cell culture, with a specific activity value of approximately 18 Umg(-1). The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory.

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 (P(i)) 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 1L cell culture, with a specific activity value of 80 Umg(-1).