Luiz Bezerra de Carvalho Júnior

Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives.

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

Dot enzyme-linked immunosorbent assay (dot-ELISA) for schistosomiasis diagnosis using dacron as solid-phase

Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA) for schistosomiasis and compared to indirect immunofluorescence (IMF). Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP) was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05). The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05). In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.

Standardization of the dot enzyme-lynked immunosorbent assay (dot-ELISA) for experimental plague

A dot enzyme linked immunosorbent assay (dot-ELISA) was previously developed to detect specific antibodies in rabbits sera immunized against F1A protein obtained from Yersinia pestis. This antigen was covalently linked onto the surface of dacron (polyethyleneterephthalate). Here, standard conditions are described for the optimization of this procedure: an amount of 20 ng of F1A protein was fixed onto dacron; anti-rabbit IgG peroxidase conjugate diluted 1:8,000 and 30% non-fat instant milk as blocking substance were used throughout the method. This procedure was compared with that employing nitrocellulose as solid-phase which showed to be more sensitive. However, the method based on dacron did not show false positive reactions against non-immunized rabbits sera at low antigen amount and diluted anti-IgG peroxidase conjugate.

Treatment of human acute schistosomiasis with oxamniquine induces an increase in interferon-gamma response to Schistosoma mansoni antigens

Patients with acute schistosomiasis were studied before and after oxamniquine treatment. They had been exposed to cercariae 5 to 9 weeks before, and presented compatible clinical manifestations, eosinophilia, and high levels of total IgE. Interferon-gamma (IFN-gamma) and interleukin-4 were measured by ELISA in whole blood samples under soluble egg antigen or soluble adult worm preparation stimulation. After treatment, the reduction of leukocytosis and eosinophilia were not significant, but total IgE levels decreased significantly, in contrast to IFN-gamma levels that were significantly increased. The oxamniquine treatment of acute schistosomiasis patients is followed by an improvement of a Th1 response in vitro. If this response has a protective aspect is unknown, and some investigations need to be realized.

Immunohistochemiluminescence detection: A quantitative tool in breast cancer HER-2 status evaluation

Her-2 status evaluation in breast cancer has prognostic and treatment response value but its interobserver variation among pathologists is a problem since it is not quantitatively assayed. This study presents an immunohistochemiluminescence method to quantify Her-2 in breast cancer. Anti-Her-2 antibody was conjugated to acridinium ester (AE) and used to evaluate/quantify Her-2 status in breast Invasive Ductal Carcinoma (IDC, n = 50) comparing with traditional immunohistochemistry. Anti-HER-2-AE results were expressed in Relative Lights Units (RLU) and showed to be able to distinguish and quantify the differences between the three groups of Her-2 status. 3+ Her-2 status presented the highest RLU (246,982 × 103 ± 2.061 × 103) compared to 2+ (76,146 × 103 ± 0.290 × 103), negative (27,415 × 103 ± 1.445 × 103) and normal tissues (27,064 × 103 ± 2.060). Status differences were significant between 3+ and 2+ (p = 0.0025); 2+ and negative (p = 0.0003), and +3 and +1 (p = 0.0001) beside this, normal breast control RLU was 27,064 × 103 ± 2,060 × 103, similar to negative cases. Results showed that anti-HER-2-AE conjugate was effective in breast tumors Her-2 status evaluation, allowing its quantitative establishment to consequently decrease the subjectivity in prognostic and predictive information intrinsic to this test.

Glycophenotype evaluation in cutaneous tumors using lectins labeled with acridinium ester.

Background. Tumor cells show alterations in their glycosylation patterns when compared to normal cells. Lectins can be used to evaluate these glycocode changes. Chemiluminescence assay is an effective technique for quantitative analysis of proteins, nucleic acids, and carbohydrates due to its high sensitivity, specificity, and rapid testing. Objective. To use histochemiluminescence based on lectin conjugated to acridinium ester (AE) for the investigation of glycophenotype changes in cutaneous tumors. Methods. Concanavalin A (Con A), Peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I), and Maackia amurensis agglutinin (MAA) were conjugated to acridinium ester. Biopsies of cutaneous tumors and normal skin were incubated with the lectins-AE, and chemiluminescence was quantified and expressed as Relative Light Units (RLU). Results. Actinic keratosis (AK), keratoacanthoma (KA), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) showed lower expression of α-D-glucose/mannose and α-L-fucose residues compared to normal tissue. Cutaneous tumors displayed higher expression of Gal-β(1-3)-GalNAc residues than normal tissue. AK and SCC exhibited higher expression of Neu5Ac-α(2,3)Gal residues than normal epidermis. KA and BCC showed equivalent RLU values compared to normal tissue. Conclusions. Lectin histochemiluminescence allowed quantitative assessment of the carbohydrate expression in cutaneous tissues, contributing to eliminate the subjectivity of conventional techniques used in the histopathological diagnosis.

Human T Cell Leukemia Virus Type 1 (HTLV-1) Antibodies in Healthy Populations and Renal Transplanted Patients in the North-East of Brazil

The seroprevalence of human T cell leukemia virus type 1 (HTLV‐1) infection was investigated in Brazilians (570): native inhabitants (298) and descendants from Japanese (272) living in Recife and its neighborhoods—North‐east of Brazil. Furthermore, polytransfused renal transplanted patients (54) were also examined for the serological status to this virus. The seropositivity to HTLV‐1, screened by enzyme‐linked immunosorbent assay (ELISA), was low: 1.34% for the local population and 0.73% for the descendants from Japanese. However, the seropositivity for the renal transplanted patients was found to be 11.1%. This higher value suggests that this retrovirus infection seems to be of importance in this clinical condition.

Trypsin purification using magnetic particles of azocasein-iron composite

This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources.

Ultrastructural Assessment of 2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide activity on human breast adenocarcinoma cells.

The aim of the present study was to investigate ultrastructural changes induced by (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (APHCA) treatment on human breast adenocarcinoma cancer cells MCF-7, besides the evaluation of phosphatidylserine externalization and DNA fragmentation in treated cells. Cell viability analysis demonstrated concentration and time-manner cytotoxicity. Treated MCF-7 cells did not expose phosphatidylserine residues to the external plasma membrane surface and DNA fragmentation was not visualized by electrophoresis. Light microscopy showed compromised cell density and presence of vacuolization after APHCA treatment with 60μM. Scanning and transmission electron microscopies revealed hallmarks of autophagy, namely the presence of membrane bebbling and autophagosomes, besides shrunken cells and cell debris in treated MCF-7 cells. However, more specific tests such as the quantification of mammalian autophagy proteins are necessary to determine the kind of death that is trigged by APHCA.

Optimization of Penicillium aurantiogriseum protease immobilization on magnetic nanoparticles for antioxidant peptides’ obtainment

ABSTRACT This work reports an optimization of protease from Penicillium aurantiogriseum immobilization on polyaniline-coated magnetic nanoparticles for antioxidant peptides’ obtainment derived from bovine casein. Immobilization process was optimized using a full two-level factorial design (24) followed by a response surface methodology. Using the derivative, casein was hydrolyzed uncovering its peptides that were sequenced and had antioxidant properties tested through (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (ABTS) radical scavenging and hydrogen peroxide scavenging assays. Optimal conditions for immobilization were 2 hr of immobilization, offered protein amount of 200 µg/mL, immobilization pH of 6.3 and 7.3 hr of activation. Derivative keeps over 74% of its original activity after reused five times. Free and immobilized enzyme casein hydrolysates presented similar peptide mass fingerprints, and prevalent peptides could be sequenced. Hydrolysates presented more than 2.5× higher ROS scavenging activity than nonhydrolyzed casein, which validates the immobilized protease capacity to develop casein-derived natural ingredients with potential for functional foods.