Reverse supply chain practices for construction and demolition waste in the Brazilian Amazon: a multi-stakeholder view.

Construction and demolition waste (CDW) is an environmental problem that affects all regions of the world. Particularly in the Brazilian Amazon Forest region, the volume of CDW generated almost doubled between 2007 and 2019. Indeed, despite Brazil having environmental regulations for waste management, these have been insufficient to solve the environmental problem because there is no CDW reverse supply chain (RSC) properly developed in the Amazon region. Previous studies have proposed a conceptual model of a CDW RSC but have hitherto failed to apply them against real world practice. This paper, therefore, attempts to test existing conceptual models that describe a CDW RSC against real industry practice prior to developing an applied model of a CDW RSC for the Brazilian Amazon. To modify the conceptual model for CDW RSC, qualitative data through 15 semi-structured interviews with five different types of stakeholders of the Amazonian CDW RSC were collected and analyzed using qualitative content analysis methods using NVivo software. The proposed applied model includes present and future reverse logistics (RL) practices, and strategies and tasks necessary for the implementation of a CDW RSC in the city of Belém of Pará, in the Brazilian Amazon. Findings reveal that several overlooked problems, particularly the limitations of the existing legal framework in Brazil, are not enough to promote a robust CDW RSC. This is perhaps the first study to examine CDW RSC in the Amazonian rainforest. Arguments provided in this study highlight the necessity for an Amazonian CDW RSC that must be promoted and regulated by the government. This can be addressed by the utilizing public-private partnership (PPP) for developing a CDW RSC.

Epidermal growth factor receptor regulates fibrinolytic pathway elements in cervical cancer: functional and prognostic implications.

Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform (http://www.cbioportal.org/), cancer cell lines, and an in vivo model of immunocompromised mice to evaluate a possible cooperation between EGFR signaling, uPA, and TM expression/function in the context of cervical cancer. cBioPortal analysis revealed that EGFR, uPA, and TM are positively correlated in tumor samples of cervical cancer patients, showing a negative prognostic impact. Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.

Epidermal growth factor receptor regulates fibrinolytic pathway elements in cervical cancer: functional and prognostic implications

Epidermal growth factor receptor (EGFR) signaling and components of the fibrinolytic system, including urokinase-type plasminogen activator (uPA) and thrombomodulin (TM), have been implicated in tumor progression. In the present study, we employed cBioPortal platform (http://www.cbioportal.org/), cancer cell lines, and an in vivo model of immunocompromised mice to evaluate a possible cooperation between EGFR signaling, uPA, and TM expression/function in the context of cervical cancer. cBioPortal analysis revealed that EGFR, uPA, and TM are positively correlated in tumor samples of cervical cancer patients, showing a negative prognostic impact. Aggressive human cervical cancer cells (CASKI) presented higher gene expression levels of EGFR, uPA, and TM compared to its less aggressive counterpart (C-33A cells). EGFR induces uPA expression in CASKI cells through both PI3K-Akt and MEK1/2-ERK1/2 downstream effectors, whereas TM expression induced by EGFR was dependent on PI3K/Akt signaling alone. uPA induced cell-morphology modifications and cell migration in an EGFR-dependent and -independent manner, respectively. Finally, treatment with cetuximab reduced in vivo CASKI xenografted-tumor growth in nude mice, and decreased intratumoral uPA expression, while TM expression was unaltered. In conclusion, we showed that EGFR signaling regulated expression of the fibrinolytic system component uPA in both in vitro and in vivo settings, while uPA also participated in cell-morphology modifications and migration in a human cervical cancer model.

Prognostic factors associated with overall survival in patients with oral cavity squamous cell carcinoma.

BACKGROUND: Low socioeconomic status, increasing age, and poor lifestyle behaviors are associated with poor survival in patients with oral cavity squamous cell carcinoma (OCSCC). To determine the overall survival (OS) and the risk of OCSCC death by tumor subsite. MATERIAL AND METHODS: A retrospective cohort study of OCSCC patients diagnosed from 2007 to 2009 and treated at a single cancer center in Rio de Janeiro, Brazil. Patient information was obtained from the Hospital Cancer Registry (HCR) database and complemented by individual search of physical and electronic medical records. Descriptive statistics of population characteristics were computed. OS was estimated using the Kaplan-Meier method. Univariate and multivariate Cox proportional hazards regression analyses were used to estimate the risk of death by tumor subsite. RESULTS: Seven hundred and three patients with OCSCC were identified. Most patients were men (77.4%) with low levels of education (67.5%), who drank (73.9%) and smoked (79.7%). The most prevalent tumor site was the tongue (45.4%), 73.4% of patients had advanced (clinical stage III or IV) OCSCC at diagnosis and 74.1% died during follow-up. For the entire cohort, the OS was 39.1% at two years and 27.9% at five years. The median survival time was 1.4 years (95%CI: 1.2‒1.5). Non-operative treatment (HR: 3.11; 95%CI: 2.26‒4.29; p<0.001), advanced stage (HR 2.14; 95%CI 1.68-2.74; p<0.001), and age >60 years at diagnosis (HR: 1.37; 95%CI: 1.15‒1.64; p<0.001) were independently associated with the risk of death. However, these factors varied by tumour subsite. CONCLUSION: Analysis of specific subsites of the oral cavity revealed substantial differences in prognostic factors associated with poor survival in OCSCC.

Analysis of the association of IL4 polymorphisms with orthodontic mini-implant loss.

The aim of this study was to investigate the association of clinical characteristics and IL4 tag single nucleotide polymorphisms (SNPs; rs2227284 and rs2243268) with orthodontic mini-implant (MI) failure. The sample included 135 subjects of both sexes, mean age 48.7±10years (range 20-76years): 104 in the control group (patients without any MI loss) and 31 in the study group (patients presenting ≥1 MI loss). Genotypes were determined by real-time PCR. Bivariate and multivariate analyses were performed (P<0.05). No association was found between the selected tag SNPs and MI loss. The C allele of the IL4 rs2243268 polymorphism in the recessive model was more frequent in patients who had fewer MIs installed (≤2 vs. >2; P=0.043, odds ratio 0.65, 95% confidence interval 0.58-0.74). On multivariate analysis, smoking habit was significantly associated with the group with multiple MIs installed (P=0.036), however the significance of the association with rs2243268 was not maintained. No association was found between the socio-demographic, smoking, or genetic factors studied and MI loss. This study supports the interaction between host and environmental factors and its influence on susceptibility to orthodontic MI failure.

Perturbative measurement of magnetoresistance.

In this paper, we report the development of a novel technique in which the magnetoresistance of nanostructures is perturbatively measured by transversally modulating the DC magnetic field. It measures the electrical transport counterpart of the transverse magnetic AC-susceptibility. The technique was developed in a conventional four-probe configuration in which a small DC current flows through the sample and a small transverse AC-field perturbates the equilibrium position of the sample magnetization. Lock-in detection, in-phase with the AC-perturbation, is used to measure the voltage signal between the two inner electrodes of the four-probe station. We successfully demonstrated that this signal is proportional to the product of the first derivative of sample resistance with respect to the equilibrium position of the magnetization times the second derivative of the energy with respect to the equilibrium position of the magnetization. These dependences add new features to the technique investigated here that were not captured by the investigations previously reported on the same subject. To show the effective use of the technique, we discuss its application in measuring magnetic properties of thin magnetic films in the non-saturated regime.

Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer.

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

Do HLA class II genes protect against pulmonary tuberculosis? A systematic review and meta-analysis.

Pulmonary tuberculosis (PTB) develops by a complex combination of environmental, immunological and socioeconomic factors and genetic susceptibility. The human leukocyte antigen (HLA) is the most polymorphic biological system and plays an essential role in the immune response against PTB. The aim of this study was to carry out a systematic review and meta-analysis evaluating the relationship between HLA-DRB1, HLA-DQB1 and HLA-DQA1 gene polymorphisms as possible risk or protective factors for PTB. A systematic search of the PubMed and Scopus databases was conducted following the guidelines described in the PRISMA statement. Fifty-six alleles were included in the meta-analysis. In the total pooled results, HLA-DRB1*08:03 (OR 1.95, CI 1.29-2.96), HLA-DQB1*06:01 (OR 1.78, CI 1.39-2.28), HLA-DQB1*06:09 (OR 2.27, 95 % CI 1.04-4.96) and HLA-DQA1*01:01 (OR 2.12, CI 1.11-4.03) genes were related to higher susceptibility to PTB. Conversely, the presence of the genes HLA-DRB1*07:01 (OR 0.74, CI 0.56-0.97), HLA-DQB1*03:01 (OR 0.77, CI 0.61-0.97), HLA-DQB1*04:02 (OR 0.57, CI 0.39-0.83), HLA-DQA1*04:01 (OR 0.50, CI 0.26-0.95) and HLA-DQA1*05:01 (OR 0.66, CI 0.48-0.92) demonstrated protection against PTB. In an analysis by ethnic subgroups, we found more genetic associations in Caucasians than in Asians. These findings suggest that HLAs may be used as markers for acquisition and development of PTB. To strengthen PTB susceptibility/resistance, we recommend further multicentric studies in different geographic regions, with certainty of controls' exposure to M. tuberculosis by use of marker of latent or active PTB, with analysis stratified by ethnic groups, with descriptions of specific alleles and carrying out immunological functionality tests.

Exploiting HPV-Induced Carcinogenesis for a Rational Drug Development in Cervical Cancer.

Cervical carcinomas are almost universally associated with high-risk human papillomavirus (HPV) infections, and are a leading cause of cancer death in women worldwide. Since the late 1990s, when a spate of studies reported the benefit of cisplatin-based chemotherapy, there had been a dearth of clinical trials in cervical cancer (CC). More effective therapies in locally advanced and recurrent or metastatic CC are an urgent clinical need. In the era of molecular oncology one should look beyond conventional chemoradiation and chemotherapy for locally advanced and advanced CC. The fact that the initiating oncogenic insult, infection with a high-risk HPV and viral oncoprotein expression is common to almost all CC offers unique opportunities for disease control. Diverse biologic pathways with an implication in the development and progression of CC are being explored. For the first time, increase in overall survival has recently been obtained for advanced CC patients with a target drug, the antiangiogenic agent bevacizumab, and durable complete responses after HPV-targeted adoptive T cell therapy in metastatic CC patients were achieved. In this review, we will summarize molecular aspects of HPV infection focusing on potential targets to stop the carcinogenic process, present updated drug development data, and discuss challenges and prospects for the future.

Outcome of orthodontic mini-implant loss in relation to interleukin 6 polymorphisms.

Mini-implants (MIs) are used increasingly for orthodontic anchorage and their success may require some osseointegration, which is affected by the underlying host immune-inflammatory response. Interleukin 6 (IL-6) is a cytokine expressed during the host response after a trauma or infection. The aim of this study was to investigate the association of clinical characteristics and IL6 tag single nucleotide polymorphisms (which capture the information of the whole gene in terms of genetic variability) with the loss of MIs for orthodontic anchorage. A total of 487 patients were treated with orthodontic MIs between 2004 and 2010. After the application of inclusion and exclusion criteria, the sample comprised 104 patients with one or more MIs that had been in function for at least 6 months with no loss, and 31 patients who had lost one or more MIs. Allele A of rs2069843 and allele T of rs2069849 were suggestively associated with the loss of MIs for orthodontic anchorage and were in complete linkage disequilibrium, which means that one of them is sufficient to capture the same information. The location of installation (mandible) and the number of MIs installed per patient were also associated with the loss of MIs.

Response of lactating cows to live yeast supplementation during summer.

Dairy cows experiencing heat stress have reduced intake and increased reliance on glucose, making feeding strategies capable of improving diet digestibility plausible for improving postrumen nutrient flow and performance. The effect of yeast on digestion and performance of lactating cows during the warm summer months of southeastern Brazil was evaluated. Cows were individually fed in tie stalls and temperature-humidity index was above 68 during 75.6% of the experiment. Twenty-eight Holstein cows (207±87 d in milk) received a standard diet for 14 d and then a treatment for 70 d, in a covariate-adjusted, randomized block design with repeated measures over time. Treatments were yeast (Saccharomyces cerevisiae) or control. Yeast was top dressed to the diet in the morning, equivalent to 25×10(10) cfu of live cells and 5×10(10) cfu of dead cells. The diet contained corn silage (37.7%), Tifton silage (7.1%), raw soybeans (4.1%), soybean meal (16.5%), finely ground corn (20.7%), and citrus pulp (11.9%). Yeast increased milk (26.7 vs. 25.4 kg/d) and solids yield (3.06 vs. 2.92 kg/d), especially lactose. Response in milk yield was consistent over time and started at d 5. The daily intake of digestible OM, total-tract digestibility of nutrients, urinary allantoin excretion, chewing pattern throughout the day, and dry matter intake did not respond to yeast. A trend was observed for increased plasma glucose with yeast (62.9 vs. 57.3mg/dL), lowered respiratory frequency (48 vs. 56 breaths/min), and increased plasma niacin content (1.31 vs. 1.22 µg/mL), though cows had similar rectal temperature. Ruminal lactate and butyrate as proportions of ruminal organic acids were reduced by yeast, but no effects on other organic acids, ruminal pH, or protozoa content were detected. Plasma urea N over 24h was increased by yeast. On d 72 to 74, citrus pulp was abruptly replaced with finely ground corn to induce acidosis. The increased load of starch increased dry matter intake between 0700 and 1300 h, jugular blood partial pressure of CO2, HCO3-, and base excess, and decreased blood pH for both treatments. The yeast treatment had a higher blood pH compared with the control, 7.34, and 7.31, respectively. Yeast supplementation improved lactation performance of dairy cows under heat stress. Improvement in lactation performance apparently involved the regulation of body homeothermia, rather than improved digestibility.

Silencing of P-glycoprotein increases mortality in temephos-treated Aedes aegypti larvae.

Re-emergence of vector-borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P-glycoproteins (P-gps) are ATP-dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P-glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P-gps. The basal ATPase activity of ATP-binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P-gp (AaegP-gp) gene expression was detected in temephos-treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP-gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)-mediated silencing of the AaegP-gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide-induced P-gp expression can be involved in the modulation of insecticide efflux.

Monocyte activation is a feature of common variable immunodeficiency irrespective of plasma lipopolysaccharide levels.

Common variable immunodeficiency disorders (CVID), the most frequent cause of symptomatic primary immunodeficiency, are defined by impaired antibody production. Notwithstanding, T cell activation and granulomatous manifestations represent the main causes of CVID morbidity even in patients receiving immunoglobulin (Ig) G replacement therapy. Additionally, gut pathology is a frequent feature of CVID. In this study, we investigated monocyte imbalances and their possible relationship with increased microbial translocation in CVID patients. Monocyte subsets were defined according to CD14 and CD16 expression levels and evaluated in terms of human leucocyte antigen D-related (HLA-DR), CD86 and programmed death-1 molecule ligand 1 (PD-L1) expression by flow cytometry, in parallel with the quantification of plasma lipopolysaccharide (LPS) and serum levels of soluble CD14 (sCD14), LPS-binding protein (LBP) and anti-LPS antibodies. CVID patients (n=31) featured significantly increased levels of serum sCD14 and an expansion of CD14(bright) CD16(+) monocytes in direct correlation with T cell and B cell activation, the latter illustrated by the frequency of the CD21(low) CD38(low) subset. Such alterations were not observed in patients lacking B cells due to congenital agammaglobulinaemia (n=4). Moreover, we found no significant increase in circulating LPS or LBP levels in CVID patients, together with a relative preservation of serum anti-LPS antibodies, in agreement with their presence in commercial IgG preparations. In conclusion, CVID was associated with monocyte imbalances that correlated directly with T cell activation markers and with B cell imbalances, without an association with plasma LPS levels. The heightened monocyte activated state observed in CVID may represent an important target for complementary therapeutic strategies.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants.

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70 percent increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

A distributed computation of Interpro Pfam, PROSITE and ProDom for protein annotation

Interpro is a widely used tool for protein annotation in genome sequencing projects, demanding a large amount of computation and representing a huge time-consuming step. We present a strategy to execute programs using databases Pfam, PROSITE and ProDom of Interpro in a distributed environment using a Java-based messaging system. We developed a two-layer scheduling architecture of the distributed infrastructure. Then, we made experiments and analyzed the results. Our distributed system gave much better results than Interpro Pfam, PROSITE and ProDom running in a centralized platform. This approach seems to be appropriate and promising for highly demanding computational tools used for biological applications.

Retrospective study of a plague outbreak by multiplex-PCR.

AIMS: To determine the effectiveness of multiplex-PCR in Yersinia pestis identification in samples preserved in Cary & Blair medium and to evaluate if this technique would uncover Y. pestis-positives among culture-negative samples. METHODS AND RESULTS: Multiplex-PCR was used to detect Y. pestis in Cary & Blair preserved bubo aspirates from experimentally infected guinea pigs and to re-analyze samples from a plague outbreak after prolonged storage in Cary & Blair. Variation in the target genes amplification was observed over time. CONCLUSIONS: Multiplex-PCR proved to be more effective than culture for plague diagnosis, both for old and recent samples. This technique would be a valuable tool for the plague control programme. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex-PCR technique can be useful for the detection and characterization of Y. pestis even when the bacteria are no longer viable and when culture diagnosis has been hampered by the growth of contaminants.

Microfluorimetric analysis of a purinergic receptor (P2X7) in GH4C1 rat pituitary cells: effects of a bioactive substance produced by Pfiesteria piscicida.

Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH4C1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH4C1 rat pituitary cells express functional P2X7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2'-3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH4C1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH4C1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH4C1 cells express a purinoceptor with characteristics consistent with the P2X7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP.

Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species.

Crithidia oncopelti, Crithidia deanei and Crithidia desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Gelatin-SDS-PAGE analysis was used to characterize cell-associated and extracellular proteinases in these organisms. Our survey indicates that the proteolytic profiles of C. deanei and C. desouzai are identical; that C. oncopelti displays a distinct zymogram; and that species naturally lacking endosymbionts have a more complex extracellular proteolytic activity, which illustrates the heterogeneity of this genus. This is the first report on the presence of cysteine proteinases in the culture supernatant of monoxenic trypanosomatids, and by the use of wild and aposymbiotic strains from C. deanei we also demonstrated that the prokaryote endosymbiont somehow alters quantitatively the expression of extracellular proteinases in this trypanosomatid.