Ralph Gruppi Thomé

Dual roles for autophagy during follicular atresia in fish ovary

Autophagy, a highly conserved catabolic program for degrading proteins and organelles, is essential for cell and tissue homeostasis. Primarily, this process has a cytoprotective role under nutrient deprivation, but several stress stimuli can induce autophagy and, thus, distinct programmed cell death (PCD) pathways can be actived when stress is not abolished. Fish ovaries are a suitable experimental model system for studying the mechanisms of PCD due to the presence of postovulatory and atretic (i.e., non-ovulated) follicles, which follow different routes after spawning. Apoptosis of the follicular cells is the major mechanism responsible for the rapid resorption of the postovulatory follicles. Recently, we investigated the contribution of PCD during follicular atresia in two species of freshwater fish. In contrast to mammals, this study revealed that follicular apoptosis is not a major process for deletion of follicular cells in atretic follicles. Furthermore, we detected autophagic vacuoles containing degenerating organelles increasing through follicular atresia in both species. In this addendum, we propose a hypothesis for follicular cell removal during ovarian regression in oviparous fish. In this model, autophagy could have dual roles in follicular atresia. Thus, fish ovaries after breeding are suitable models for studying the interactions among the different cell death pathways.

Evaluation of healing wound and genotoxicity potentials from extracts hydroalcoholic of Plantago major and Siparuna guianensis

Despite the large use of the Plantago major and Siparuna guianensis in traditional medicine, there are no studies demonstrating the effectiveness from extracts of these plants in the healing process by the present methodology. This study reported the effects and toxicity of the P. major and S. guianensis extracts in the wound healing compared with a commercial product used in Brazil by macroscopic and microscopic analysis. Following injury in cervical dorsal area of the mice, the extract from P. major and S. guianensis and ointment was applied after an injury in cervical dorsal area of the mice. Wound healing rates were calculated at 4, 9, 15 and 21 d after the wounding, and tissues were obtained on the ninth day for histological analysis. Moreover, mutagenic assay of extracts was performed. Mutagenicity studies carried out with plant extracts showed not mutagenic with or without metabolic activations. Reduction of the wound area occurred earlier in mice treated with P. major and control treatment. On the 15th day, the complete wound closure occurred in P. major-treated wounds. Throughout ointment and S. guianensis treatment it was not observed the wound closured. Microscopic analyses of the wound, on the ninth day, showed the more efficient formation of the neoepithelium and skin appendages in animals treated with S. guianensis and P. major, while ointment treatment presented no re-epithelialization and absent skin appendages in wound. Thus, P. major extract showed good effects on wound healing processes rendering it a promising candidate for the treatment of wounds what also justified its traditional usage in wound treatment.

Relationship between bcl-2, bax, beclin-1, and cathepsin-D proteins during postovulatory follicular regression in fish ovary

In fish ovaries, postovulatory follicles (POFs) are key biomarkers of breeding and provide an interesting model for studying the relationship between autophagy and apoptosis. In this study, we investigated the immunohistochemical expression of autophagic and apoptotic proteins to improve the knowledge on the mechanisms regulating ovarian remodeling after spawning. Females from three neotropical fish species kept in captivity were submitted to hormonal induction. After ova stripping, ovarian sections were sampled daily until 5 days postspawning (dps). Similar events of POF regression were detected by histology, terminal transferase-mediated dUTP nick-end labeling (TUNEL), and electron microscopy in the three species: follicular cells hypertrophy, progressive disintegration of the basement membrane, gradual closing of the follicular lumen, theca thickening, and formation of large autophagic vacuoles preceding apoptosis of the follicular cells. Autophagic and apoptotic proteins were assessed by immunohistochemistry. Morphometric analysis of the immunolabeling revealed a more intense reaction for bcl-2 and beclin-1 (BECN1) in POFs at 0 to 1 dps and for bax at 2 to 3 dps (P < 0.001), the later period being the peak of apoptosis of the follicular cells. The immunostaining for cathepsin-D was more elevated until 2 to 3 dps and decreased significantly at 4 to 5 dps, when the POFs were in late stage of regression. Double labeling for BECN1 and caspase-3 indicated a shift in the relationship between autophagy and apoptosis at 2 to 3 dps, a critical period in determining the fate of follicular cells in POFs. Together, these results indicate that the bcl-2 family, BECN1, and cathepsin-D can be involved in the regulation of ovarian remodeling in teleost fish.

Comparative histology in the liver and spleen of three species of freshwater teleost

Nos descrevemos e comparamos a histologia do figado e do baco de Geophagus brasiliensis (Perciformes), Hypostomus francisci (Siluriformes) e Hoplias aff. malabaricus (Characiformes), peixes neotropicais de agua doce. Em G. brasiliensis e H. aff. malabaricus os hepatocitos organizaram-se na forma tubular enquanto que em H. francisci eles apresentaram-se como cordoes celulares. Em todas as especies, os hepatocitos apresentaram glicogenio, mas em G. brasiliensis e H. aff. malabaricus, eles mostraram forte marcacao para hemossiderina no citoplasma. Em G. brasiliensis e H. aff. malabaricus, centros melanomacrofagicos (CMMs) foram associados a estruturas hepaticas e somente em G. brasiliensis foi observado pâncreas exocrino intrahepatico. O baco, em todas as especies, foi caracterizado pela polpa vermelha e branca sem limites entre as duas regioes, mas somente em H. francisci foi registrado uma organizacao nodular no parenquima esplenico. G. brasiliensiseH. aff. malabaricusapresentaram na polpa branca CMMs associados principalmente a elipsoides. Alem disso, nos observamos CMMs grandes no baco em relacao ao figado de G. brasiliensis e de H. aff. malabaricus. No figado, valores altos de fibras reticulares e colageno foram observado em G. brasiliensis. No baco, valores altos de fibras reticulares e colageno foram registrados em H. aff. malabaricuseH. francisci, respectivamente. Diferencas histologicas confirmam a hipotese que a distância filogenetica esta refletida no figado e no baco.

Immunohistochemical Expression of Collagens in the Skin of Horses Treated with Leukocyte-Poor Platelet-Rich Plasma

This study evaluated the immunohistochemical expression of type I (COL I) and III (COL III) collagens during the healing process of skin treated with leukocyte-poor platelet-rich plasma (LP-PRP). Seven healthy gelding crossbred horses aged 16 to 17 years were used. Two rectangle-shaped wounds were created surgically in the right and left gluteal regions. Twelve hours after wound induction, 0.5 mL of the LP-PRP was administered in each edge of the wounds of one of the gluteal regions. The contralateral region was used as control (CG). Three samples were obtained: after wound induction (T0), 14 days (T1) of healing process, and after complete closure of the skin (T2). The normal skin (T0) showed strong staining for type III and I collagen in papillary and reticular dermis, respectively. In the scar of the treated group, COL III showed important (p < 0.05) increase in immunoreaction in T2 compared with T1. The administration of a single dose of LP-PRP 12 h after induction of wound in horses does not influence formation of collagens I and III. However, the intense labeling for COL III suggests that the tissue was still weak during the macroscopic closure of the wound, demonstrating that healing was not completely finished.

Assessment of fish assemblage in an urban system, Itapecerica River, upper São Francisco River basin, Divinópolis, Minas Gerais, Brazil

Despite the economic and ecological importance of the Itapecerica River, there is no information about the local fish fauna. The aim of the present study was to provide a list of fish species in Itapecerica River, a tributary of the upper Sao Francisco River basin, inside an urban area. The specimens were collected quarterly from May 2010 to February 2012, using casting nets, trawl net and gillnets. We listed 29 species belonging to 21 genera of 14 families and 4 orders. Characiformes were represented by 16 species, Siluriformes by nine, Gymnotiformes and Perciformes by two each. Three alien species and five migratory fish were recorded. The low number of species found in this study in relation to Sao Francisco basin, could be reflecting the high level of degradation of waters in the Itapecerica River.

Proliferation, survival and cell death in fish gills remodeling: From injury to recovery

Abstract Pollutants found dispersed in water can cause irritations on the gills, challenge the immune system and prejudice the welfare of the fish. Here we investigated molecules linked to proliferation, survival, and cell death, as well as inflammatory and vascular control, in a model of fish gill remodeling, from injury to recovery. We assessed the gill histology and immunohistochemistry for PCNA, iNOS, HSP70, and Bax in Hypostomus francisci obtained from a river subjected to chronic anthropic influences and then after they were placed in water of good quality. A total of 30 H. francisci adult individuals were collected and distributed into two groups: euthanized on the day of capture (group 1) and maintained for 30 days in an aquarium (group 2). In all the fish from group 1, the primary and secondary lamellae showed hypertrophy of the respiratory epithelium, lamellar fusion, lifting of the epithelium, aneurysm, hyperemia, and vascular congestion. On the other hand, in all the fish from group 2, restoration of gill integrity was observed, and the primary and secondary lamellae showed a simple epithelium, absence of lamellar fusion, hypertrophy, and aneurysm. Gills of fish from group 1 had higher frequency of cells immunopositive for PCNA, iNOS, HSP70, and Bax than those of fish from group 2 (p < 0.05). The molecular and cellular mechanisms from injury to recovery were proposed, with a balance between survival and cell death signals being essential for determining the gill structure. In addition, the findings indicate that recovery of the structural organization of gills is possible if fishes are maintained in good‐quality water, indicating the importance of the conservation of aquatic environments. HighlightsGill tissue changes can be used as biomarker of non‐point source of pollution.In gills injury, cell proliferation and vascular changes are the primary response.Recovery gill tissue is possible in animals maintained in a good quality water.A balance between survival or death, and inflammatory signs being essential for the gill recovery.

Biological variables of Hypostomus francisci (Siluriformes: Loricariidae) from Itapecerica River, Minas Gerais State, Brazil

Herein we determine for the first time the reproduction parameters and population structure of Hypostomus francisci in the Itapecerica River, São Francisco Basin. A total of 250 specimens was captured quarterly between March 2010 and February 2012. Body weight, total length and weight of the gonads were obtained in the laboratory. Gonad samples were submitted to histological and histochemical techniques. Females with spawning capable ovaries were used to determine the fecundity and relative fecundity. Sex ratio with 1:1.01 (female:male) was observed. Males were more numerous than females for individuals smaller than 170 mm, however the number of females was significantly greater for specimens larger than 330 mm. The length-weight relationship estimated for H. francisci indicates negative-allometric growth. Females spawning capable were observed mostly in November-December-January. Two cohorts of oocytes at a determined time evidencing the development type group-synchronic. The eggs reaching 3.4 mm and the fecundity ranged from 312-1,460 oocytes with an average of 585.81 ± 337.43 oocytes per female. The reproductive parameters and population structure of H. francisci from Itapecerica River suggested that this species showed singular reproductive tactics among congeners.

Synthesis, cytotoxic activity, and mode of action of new Santacruzamate A analogs

Breast and ovarian cancer are the most common cancers in women. Available cancer treatments, in general, have limited efficacy and frequent, undesirable side effects. Recently, scientists have focused on searching for new epigenetic modulators such as inhibitors of DNA methyltransferases and histone deacetylases (HDACs), with novel properties and selectivity. We report the synthesis of seven new analogs of Santacruzamate A. Molecular modeling showed that compounds 3–9 presented the best binding energies (kcal/mol) against HDAC4 compared to that of crystallographic ligand. The compounds were evaluated against MCF-7 and MDA-MB-231 (breast cancer), TOV-21G (ovarian adenocarcinoma), and WI-26VA4 (non-tumor lung fibroblasts) cells. Compound 5, the most potent and selective of the series, exhibited remarkably enhanced anticancer potency, with IC50 values for the tumor cells of 24.3–44.93 μM, compared with that of etoposide (12–18.57 μM) and doxorubicin (2.1–4.37 μM). Further investigation showed that compound 5 could promote DNA damage, increase the activity of caspases-3 and -9, and upregulate mRNA levels of p21, TP53, and BAK, suggesting apoptotic cell death of the tumor cells via the intrinsic pathway. This study demonstrated that synthetic analogs of santacruzamate A with zinc-linked groups are effective for improving both HDAC inhibition and antitumor activity.

21‑Benzylidene digoxin, a novel digoxin hemi-synthetic derivative, presents an anti-inflammatory activity through inhibition of edema, tumour necrosis factor alpha production, inducible nitric oxide synthase expression and leucocyte migration

&NA; Recent findings have demonstrated new therapeutic functions of cardiotonic steroids, a process that is termed drug repositioning. Despite the confirmed anti‐inflammatory effects of cardiotonic steroids, their clinical use has been discouraged due to toxicity related to inhibition of the Na+/K+ ATPase. A novel synthetic compound derived from digoxin, 21‐benzylidene digoxin (21‐BD), does not inhibit this enzyme. Herein, we evaluated the anti‐inflammatory and antinociceptive effects and acute toxicity of 21‐BD. Murine (Swiss mice) models of paw oedema induced by carrageenan, acetic acid‐induced abdominal writhing, and formalin and acute toxicity tests were used. Oral administration of 21‐BD (0.3 mg/kg) showed a significant and prolonged inhibition of paw oedema. Histological analysis demonstrated a reduction in inflammatory cells and expression of inducible nitric oxide synthase (iNOS) in footpads 6 h after administration of carrageenan. 21‐BD (0.3 mg/kg) also reduced the levels of tumour necrosis factor (TNF)‐&agr; 2 and 4 h after carrageenan. 21‐BD demonstrated antinociceptive activity, inhibiting abdominal writhes at all tested doses. However, in the formalin test, 21‐BD did not present antinociceptive activity. In the acute toxicity test, 21‐BD did not cause symptoms of toxicity or mortality. The present study demonstrated, for the first time, that 21‐BD is safe and exhibits a marked anti‐inflammatory activity in acute local inflammation. This effect might be a consequence of its ability to inhibit the release of the PMN leucocyte‐derived mediators, including TNF‐&agr;, and iNOS expression as well as its inhibitory effect on oedema and PMN leucocyte infiltration.