Developmental staging table of the green iguana

Embryonic staging tables provide information to standardize embryological investigations and to subsidize discussions about evolution. We have established a developmental staging table for Iguana iguana iguana. The sample was composed of 142 embryos, incubated at a constant temperature and collected at regular intervals. Morphological features as pharyngeal arches, craniofacial structures, eyes, limbs, claws, pigmentation, scales and egg tooth were evaluated to determine development stages. The normal staging table includes 17 stages from oviposition to hatching, based on chronology and morphological external features. Stages from 1 to 27 occur before oviposition. Stage 28 was the first described, because all embryos presented limb bud anlage, key feature of the previous stage. We used pharyngeal arches and limb buds to describe the first stages; claws, genital papilla and scales to describe the middle stages; and pigmentation, size and egg tooth to describe the last stages. Incubation lasted approximately 2 months in a controlled environment. The results were similar to the data from other lizards, confirming the embryonic conservative pattern of the group.     

Structural characterization of cationic DODAB bilayers containing C24:1 β-glucosylceramide

The effect of 5 mol%, 9 mol%, and 16 mol% of C24:1 β-glucosylceramide (βGlcCer) on the structure of cationic DODAB bilayers was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy and fluorescence microscopy. βGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed in fluorescence microscopy or ESR spectra. The latter showed that βGlcCer destabilized the gel phase of DODAB bilayers by decreasing the gel phase packing. As a consequence, βGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB bilayers, as seen in DSC thermograms. ESR spectra also showed that βGlcCer induced an increase in DODAB fluid phase order and/or rigidity. Despite their different structures, a similar effect of loosening the gel phase packing and turning the fluid phase more rigid/organized has also been observed when low molar fractions of cholesterol were incorporated in DODAB bilayers. The structural characterization of mixed membranes made of cationic lipids and glucosylceramides may be important for developing novel immunotherapeutic tools such as vaccine adjuvants.     

Nucleus behavior during the closed mitosis of Tritrichomonas foetus

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.     

Antiproliferative effects of several compounds isolated from Amburana cearensis A. C. Smith

Amburana cearensis a common tree found in Northeastern Brazil is widely used in folk medicine. The present work evaluated the cytotoxicity of kaempferol, isokaempferide, amburoside A and protocatechuic acid isolated from the ethanol extract of the trunk bark of A. cearensis. The compounds were tested for their cytotoxicity on the sea urchin egg development, hemolysis assay and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay using tumor cell lines. Isokaempferide and kaempferol, but not amburoside A and protocatechuic acid, inhibited the sea urchin egg development as well as tumor cell lines, but in this assay isokaempferide was more potent than kaempferol. Protocatechuic acid was the only compound able to induce hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of kaempferol and isokaempeferide was not related to membrane damage.     

Cell-Associated and Extracellular Proteinases in Blastocrithidia culicis: Influence of Growth Conditions

The proteinase profile of Blastocrithidia culicis was analyzed, as well as how different growth conditions influenced its expression by gelatin-SDS-PAGE and the use of specific proteinase inhibitors. Multiple cell-associated proteinases with molecular masses corresponding to 33, 55, 60 kDa (cysteine proteinases) and 77, 80, 90, and 108 kDa (metalloproteinases) were detected using these methods. All the metalloproteinases identified were partitioned into the detergent phase after Triton X-114 extract, suggesting that they are membrane-bound, while all cysteine proteinases were partitioned into the aqueous phase. The proteolytic zymograms were similar when three different media were used for variable times of incubation. However, few quantitative and qualitative changes were observed. The secreted proteinase profile showed an heterogeneous pattern of enzymatic activities whose expression was dependent on time of growth and medium composition. However, the extracellular proteinase activities were abolished by 1,10-phenanthroline, suggesting that all of them are zinc-metalloproteinases. Received: 25 October 2000 / Accepted: 10 January 2001     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Application of an aqueous two‐phase micellar system to extract bromelain from pineapple (Ananas comosus) peel waste and analysis of bromelain stability in cosmetic formulations

Bromelain is a set of proteolytic enzymes found in pineapple (Ananas comosus) tissues such as stem, fruit and leaves. Because of its proteolytic activity, bromelain has potential applications in the cosmetic, pharmaceutical, and food industries. The present study focused on the recovery of bromelain from pineapple peel by liquid–liquid extraction in aqueous two‐phase micellar systems (ATPMS), using Triton X‐114 (TX‐114) and McIlvaine buffer, in the absence and presence of electrolytes CaCl₂ and KI; the cloud points of the generated extraction systems were studied by plotting binodal curves. Based on the cloud points, three temperatures were selected for extraction: 30, 33, and 36°C for systems in the absence of salts; 40, 43, and 46°C in the presence of KI; 24, 27, and 30°C in the presence of CaCl₂. Total protein and enzymatic activities were analyzed to monitor bromelain. Employing the ATPMS chosen for extraction (0.5 M KI with 3% TX‐114, at pH 6.0, at 40°C), the bromelain extract stability was assessed after incorporation into three cosmetic bases: an anhydrous gel, a cream, and a cream‐gel formulation. The cream‐gel formulation presented as the most appropriate base to convey bromelain, and its optimal storage conditions were found to be 4.0 ± 0.5°C. The selected ATPMS enabled the extraction of a biomolecule with high added value from waste lined‐up in a cosmetic formulation, allowing for exploration of further cosmetic potential. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:937–945, 2015     

Characterization of heme as activator of Toll-like receptor 4

Heme is an ancient and ubiquitous molecule present in organisms of all kingdoms, composed of an atom of iron linked to four ligand groups of porphyrin. A high amount of free heme, a potential amplifier of the inflammatory response, is a characteristic feature of diseases with increased hemolysis or extensive cell damage. Here we demonstrate that heme, but not its analogs/precursors, induced tumor necrosis factor-alpha (TNF-alpha) secretion by macrophages dependently on MyD88, TLR4, and CD14. The activation of TLR4 by heme is exquisitely strict, requiring its coordinated iron and the vinyl groups of the porphyrin ring. Signaling of heme through TLR4 depended on an interaction distinct from the one established between TLR4 and lipopolysaccharide (LPS) since anti-TLR4/MD2 antibody or a lipid A antagonist inhibited LPS-induced TNF-alpha secretion but not heme activity. Conversely, protoporphyrin IX antagonized heme without affecting LPS-induced activation. Moreover, heme induced TNF-alpha and keratinocyte chemokine but was ineffective to induce interleukin-6, interleukin-12, and interferon-inducible protein-10 secretion or co-stimulatory molecule expression. These findings support the concept that the broad ligand specificity of TLR4 and the different activation profiles might in part reside in its ability to recognize different ligands in different binding sites. Finally, heme induced oxidative burst, neutrophil recruitment, and heme oxygenase-1 expression independently of TLR4. Thus, our results presented here reveal a previous unrecognized role of heme as an extracellular signaling molecule that affects the innate immune response through a receptor-mediated mechanism.     

Synthesis and in vitro evaluation of leishmanicidal and trypanocidal activities of N-quinolin-8-yl-arylsulfonamides

In the present paper 12 N-quinolin-8-yl-arylsulfonamides synthesized by coupling 8-aminoquinolines with various arylsulfonylchlorides were assayed in vitro against Leishmania amazonensis, Leishmania chagasi and Trypanosoma cruzi strains. This series of new compounds were found to be selective for Leishmania spp. promastigote and amastigote forms. The most active compound was the N-(8-quinolyl)-3,5-difluoro-benzenesulfonamide 10 with an IC₅₀ against L. amazonensis and L. chagasi of 2.12 and 0.45 μM, respectively. The less cytotoxic biphenyl derivative 7 was very effective against intracellular L. amazonensis with a reduction of macrophage cell infection of 82.1% at 25 μM. In addition, a copper complex 17 of an inactive ligand was readily synthesized and showed high leishmanicidal and trypanocidal activity against both extra and intracellular forms.