Pseudomonas syringae pv. tabaci in papaya seedlings

The natural occurrence of Pseudomonas syringae pv. tabaci causing leaf spot symptoms in papaya seedlings is reported. The pathogen was identified through biochemical, physiological, serological, and molecular assays and artificial inoculations in papaya plants. It was also shown that the strains were pathogenic to bean and tobacco plants. The restriction patterns obtained with Afa I, Alu I, Dde I, Hae III, Hpa II, Hinf I, Sau 3A I and Taq I of the PCR-RFLP of 16S-23S DNAr were identical to the P. s. pv. tabaci patterns. Primers corresponding to hrpL gene of P. syringae were also tested and the results grouped the papaya strains with P s. pv. tabaci. Bacterial strains were deposited at Coleção de Culturas IBSBF, Instituto Biológico, Campinas, Brazil, under access numbers 1687 and 1822.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Outbreak of autochthonous canine visceral leishmaniasis in Santa Catarina, Brazil

The present study reports the first outbreak of autochthonous canine visceral leishmaniasis in Florianópolis, Santa Catarina, southern Brazil. Following the report of two cases of CVL, the Control Center of Zoonotic Diseases conducted a serological survey by ELISA and IFAT assays in seven districts of the Santa Catarina Island. Eleven seropositive dogs of autochthonous transmission were used in the present study. Infection by Leishmania sp. was confirmed by parasitological examination of bone marrow, liver, spleen and lymph nodes, culture in Schneider's medium and PCR. Leishmania sp. isolates were characterized by PCR-RFLP and hybridization with specific probes, allowing for the identification of Leishmania infantum. Autochthonous transmission of this disease in an area with high tourist traffic presents a major public health concern and signifies the emergence of an important zoonosis in southern Brazil. Therefore, the implementation of surveillance and control measures is imperative to prevent the spread of the disease among the canine population as well as transmission to the human population.

Markers of Bone Turnover In Preclinical Development of Drugs for Skeletal Diseases

Skeletal tissue is constantly remodeled in a process where osteoclasts resorb old bone and osteoblasts form new bone. Balance in bone remodeling is related to age, gender and genetic factors, but also many skeletal diseases, such as osteoporosis and cancer-induced bone metastasis, cause imbalance in bone turnover and lead to decreased bone mass and increased fracture risk. Biochemical markers of bone turnover are surrogates for bone metabolism and may be used as indicators of the balance between bone resorption and formation. They are released during the remodeling process and can be conveniently and reliably measured from blood or urine by immunoassays. Most commonly used bone formation markers include N-terminal propeptides of type I collagen (PINP) and osteocalcin, whereas tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and C-terminal cross-linked telopeptide of type I collagen (CTX) are common resorption markers. Of these, PINP has been, until recently, the only marker not commercially available for preclinical use. To date, widespread use of bone markers is still limited due to their unclear biological significance, variability, and insufficient evidence of their prognostic value to reflect long term changes. In this study, the feasibility of bone markers as predictors of drug efficacy in preclinical osteoporosis models was elucidated. A non-radioactive PINP immunoassay for preclinical use was characterized and validated. The levels of PINP, N-terminal mid-fragment of osteocalcin, TRACP 5b and CTX were studied in preclinical osteoporosis models and the results were compared with the results obtained by traditional analysis methods such as histology, densitometry and microscopy. Changes in all bone markers at early timepoints correlated strongly with the changes observed in bone mass and bone quality parameters at the end of the study. TRACP 5b correlated strongly with the osteoclast number and CTX correlated with the osteoclast activity in both in vitro and in vivo studies. The concept “resorption index” was applied to the relation of CTX/TRACP 5b to describe the mean osteoclast activity. The index showed more substantial changes than either of the markers alone in the preclinical osteoporosis models used in this study. PINP was strongly associated with bone formation whereas osteocalcin was associated with both bone formation and resorption. These results provide novel insight into the feasibility of PINP, osteocalcin, TRACP 5b and CTX as predictors of drug efficacy in preclinical osteoporosis models. The results support clinical findings which indicate that short-term changes of these markers reflect long-term responses in bone mass and quality. Furthermore, this information may be useful when considering cost-efficient and clinically predictive drug screening and development assays for mining new drug candidates for skeletal diseases.

Switchable Lanthanide Luminescence for Detection of Biomolecules

Binary probes are oligonucleotide probe pairs that hybridize adjacently to a complementary target nucleic acid. In order to detect this hybridization, the two probes can be modified with, for example, fluorescent molecules, chemically reactive groups or nucleic acid enzymes. The benefit of this kind of binary probe based approach is that the hybridization elicits a detectable signal which is distinguishable from background noise even though unbound probes are not removed by washing before measurement. In addition, the requirement of two simultaneous binding events increases specificity. Similarly to binary oligonucleotide probes, also certain enzymes and fluorescent proteins can be divided into two parts and used in separation-free assays. Split enzyme and fluorescent protein reporters have practical applications among others as tools to investigate protein-protein interactions within living cells. In this study, a novel label technology, switchable lanthanide luminescence, was introduced and used successfully in model assays for nucleic acid and protein detection. This label technology is based on a luminescent lanthanide chelate divided into two inherently non-luminescent moieties, an ion carrier chelate and a light harvesting antenna ligand. These form a highly luminescent complex when brought into close proximity; i.e., the label moieties switch from a dark state to a luminescent state. This kind of mixed lanthanide complex has the same beneficial photophysical properties as the more typical lanthanide chelates and cryptates - sharp emission peaks, long emission lifetime enabling time-resolved measurement, and large Stokes’ shift, which minimize the background signal. Furthermore, the switchable lanthanide luminescence technique enables a homogeneous assay set-up. Here, switchable lanthanide luminescence label technology was first applied to sensitive, homogeneous, single-target nucleic acid and protein assays with picomolar detection limits and high signal to background ratios. Thereafter, a homogeneous four-plex nucleic acid array-based assay was developed. Finally, the label technology was shown to be effective in discrimination of single nucleotide mismatched targets from fully matched targets and the luminescent complex formation was analyzed more thoroughly. In conclusion, this study demonstrates that the switchable lanthanide luminescencebased label technology can be used in various homogeneous bioanalytical assays.

Cardiac Troponin Specific Autoantibodies: Analytical Tools for Exploring Their Impact on Cardiac Troponin I Testing

Cardiac troponin (cTn) I and T are the recommended biomarkers for the diagnosis and risk stratification of patients with suspected acute coronary syndrome (ACS), a major cause of cardiovascular death and disability worldwide. It has recently been demonstrated that cTn-specific autoantibodies (cTnAAb) can negatively interfere with cTnI detection by immunoassays to the extent that cTnAAb-positive patients may be falsely designated as cTnI-negative. The aim of this thesis was to develop and optimize immunoassays for the detection of both cTnI and cTnAAb, which would eventually enable exploring the clinical impact of these autoantibodies on cTnI testing and subsequent patient management. The extent of cTnAAb interference in different cTnI assay configurations and the molecular characteristics of cTnAAbs were investigated in publications I and II, respectively. The findings showed that cTnI midfragment targeting immunoassays used predominantly in clinical practice are affected by cTnAAb interference which can be circumvented by using a novel 3+1-type assay design with three capture antibodies against the N-terminus, midfragment and C-terminus and one tracer antibody against the C-terminus. The use of this assay configuration was further supported by the epitope specificity study, which showed that although the midfragment is most commonly targeted by cTnAAbs, the interference basically encompasses the whole molecule, and there may be remarkable individual variation at the affected sites. In publications III and IV, all the data obtained in previous studies were utilized to develop an improved version of an existing cTnAAb assay and a sensitive cTnI assay free of this specific analytical interference. The results of the thesis showed that approximately one in 10 patients with suspected ACS have detectable amounts of cTnAAbs in their circulation and that cTnAAbs can inhibit cTnI determination when targeted against the binding sites of assay antibodies used in its immunological detection. In the light of these observations, the risk of clinical misclassification caused by the presence of cTnAAbs remains a valid and reasonable concern. Because the titers, affinities and epitope specificities of cTnAAbs and the concentration of endogenous cTnI determine the final effect of circulating cTnAAbs, appropriately sized studies on their clinical significance are warranted. The new cTnI and cTnAAb assays could serve as analytical tools for establishing the impact of cTnAAbs on cTnI testing and also for unraveling the etiology of cTn-related autoimmune responses.

Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line

The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, São Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis), as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA) and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified.

HHV-8 infection in patients with AIDS-related Kaposi's sarcoma in Brazil

The aims of the present study were to determine the prevalence of human herpesvirus type 8 (HHV-8) in HIV-positive Brazilian patients with (HIV+/KS+) and without Kaposi's sarcoma (HIV+/KS-) using PCR and immunofluorescence assays, to assess its association with KS disease, to evaluate the performance of these tests in detecting HHV-8 infection, and to investigate the association between anti-HHV-8 antibody titers, CD4 counts and staging of KS disease. Blood samples from 66 patients, 39 HIV+/KS+ and 27 HIV+/KS-, were analyzed for HHV-8 viremia in peripheral blood mononuclear cells by PCR and HHV-8 antigenemia for latent and lytic infection by immunofluorescence assay. Positive samples for latent nuclear HHV-8 antigen (LNA) antibodies were titrated out from 1/100 to 1/409,600 dilution. Clinical information was collected from medical records and risk behavior was assessed through an interview. HHV-8 DNA sequences were detected by PCR in 74.3% of KS+ patients and in 3.7% of KS- patients. Serological assays were similar in detecting anti-LNA antibodies and anti-lytic antigens in sera from KS+ patients (79.5%) and KS- patients (18.5%). HHV-8 was associated with KS whatever the method used, i.e., PCR (odds ratio (OR) = 7.4, 95% confidence interval (CI) = 2.16-25.61) or anti-LNA and anti-lytic antibodies (OR = 17.0, 95%CI = 4.91-59.14). Among KS+ patients, HHV-8 titration levels correlated positively with CD4 counts (rho 0.48, P = 0.02), but not with KS staging. HHV-8 is involved in the development of KS in different geographic areas worldwide, as it is in Brazil, where HHV-8 is more frequent among HIV+ patients. KS severity was associated with immunodeficiency, but no correlation was found between HHV-8 antibody titers and KS staging.

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

DNA topoisomerase inhibitors: biflavonoids from Ouratea species

Topoisomerase inhibitors are agents with anticancer activity. 7"-O-Methyl-agathisflavone (I) and amentoflavone (II) are biflavonoids and were isolated from the Brazilian plants Ouratea hexasperma and O. semiserrata, respectively. These biflavonoids and the acetyl derivative of II (IIa) are inhibitors of human DNA topoisomerases I at 200 µM, as demonstrated by the relaxation assay of supercoiled DNA, and only agathisflavone (I) at 200 µM also inhibited DNA topoisomerases II-alpha, as observed by decatenation and relaxation assays. The biflavonoids showed concentration-dependent growth inhibitory activities on Ehrlich carcinoma cells in 45-h culture, assayed by a tetrazolium method, with IC50 = 24 ± 1.4 µM for I, 26 ± 1.1 µM for II and 10 ± 0.7 µM for IIa. These biflavonoids were assayed against human K562 leukemia cells in 45-h culture, but only I showed 42% growth inhibitory activity at 90 µM. Our results suggest that biflavonoids are targets for DNA topoisomerases and their cytotoxicity is dependent on tumor cell type.

Immunomodulatory properties of Alternanthera tenella Colla aqueous extracts in mice

Plants from the genus Alternanthera are thought to possess antimicrobial and antiviral properties. In Brazilian folk medicine, the aqueous extract of A. tenella Colla is used for its anti-inflammatory activity. The present study investigated the immunomodulatory property of A. tenella extract by evaluating the antibody production in male albino Swiss mice weighing 20-25 g (10 per group). The animals received standard laboratory diet and water ad libitum. The effect of A. tenella extract (5 and 50 mg/kg, ip) was evaluated in mice immunized with sheep red blood cells (SRBC 10%, ip) as T-dependent antigen, or in mice stimulated with mitogens (10 µg, Escherichia coli lipopolysaccharide, LPS, ip). The same doses (5 and 50 mg/kg, ip) of A. tenella extract were also tested for antitumor activity, using the Ehrlich ascites carcinoma as model. The results showed that 50 mg/kg A. tenella extract ip significantly enhanced IgM (64%) and IgG2a (50%) antibody production in mice treated with LPS mitogen. The same dose had no effect on IgM-specific response, whereas the 5 mg/kg treatment caused a statiscally significant reduction of anti-SRBC IgM-specific antibodies (82%). The aqueous extract of A. tenella (50 mg/kg) increased the life span (from 16 ± 1 to 25 ± 1 days) and decreased the number of viable tumor cells (59%) in mice with Ehrlich ascites carcinoma. The present findings are significant for the development of alternative, inexpensive and perhaps even safer strategies for cancer treatment.

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

Effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs on the production of reactive oxygen species by activated rat neutrophils

The release of reactive oxygen specie (ROS) by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs) on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 µM), indomethacin (12 µM), naproxen (160 µM), piroxicam (13 µM), and tenoxicam (30 µM) were incubated at 37ºC in PBS (10 mM), pH 7.4, for 30 min with rat neutrophils (1 x 10(6) cells/ml) stimulated by phorbol-12-myristate-13-acetate (100 nM). The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 ± 2% diclofenac, 90 ± 2% indomethacin, 33 ± 3% piroxicam, and 45 ± 6% tenoxicam (N = 6). For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 ± 5% and diclofenac showed amplification in the light emission of 181 ± 60% (N = 6). Using the myeloperoxidase (MPO)/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 ± 10, 45 ± 3%), indomethacin (97 ± 2, 100 ± 1%), naproxen (56 ± 8, 76 ± 3%), piroxicam (77 ± 5, 99 ± 1%), and tenoxicam (90 ± 2, 100 ± 1%), respectively (N = 3). These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.

Procoagulant properties of human MV3 melanoma cells

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

The inflammatory stimulus of a natural latex biomembrane improves healing in mice

The aim of the present study was to compare healing obtained with biomembranes with the natural healing process (sham) using biochemical and immunohistological assays. C57BL/6 mice were divided into 4 groups of 15 mice each and received different subcutaneous implants: natural latex biomembrane (NLB), denatured latex (DL), expanded polytetrafluorethylene (ePTFE), or sham. On the 2nd, 7th, and 14th days post-treatment, 5 mice per group were sacrificed and biopsied for the following measurements: oxidative stress based on malondialdehyde (MDA), myeloperoxidase (MPO) and hydrogen peroxide by the method of ferrous oxidation-xylenol orange (FOX), as well as glutathione and total proteins; histological evaluation to enumerate inflammatory cells, fibroblasts, blood vessels, and collagen, and immunohistochemical staining for inducible nitric oxide synthase, interleukin-1β, vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1). On day 2 post-treatment, NLB stimulated a dense inflammatory infiltrate mainly consisting of polymorphonuclear cells, as indicated by increased MPO (P < 0.05), but oxidative stress due to MDA was not observed until the 7th day (P < 0.05). The number of blood vessels was greater in NLB (P < 0.05) and DL (P < 0.05) mice compared to sham animals on day 14. NLB induced fibroplasia by day 14 (P < 0.05) with low expression of TGF-β1 and collagenesis. Thus, NLB significantly induced the inflammatory phase of healing mediated by oxidative stress, which appeared to influence the subsequent phases such as angiogenesis (with low expression of VEGF) and fibroplasia (independent of TGF-β1) without influencing collagenesis.