Neutrophil Extracellular Traps Identification in Tegumentary Lesions of Patients with Paracoccidioidomycosis and Different Patterns of NETs Generation In Vitro

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.     

Molecular basis of functional myogenic specification of Bona Fide multipotent adult cardiac stem cells

Ischemic Heart Disease (IHD) remains the developed world’s number one killer. The improved survival from Acute Myocardial Infarction (AMI) and the progressive aging of western population brought to an increased incidence of chronic Heart Failure (HF), which assumed epidemic proportions nowadays. Except for heart transplantation, all treatments for HF should be considered palliative because none of the current therapies can reverse myocardial degeneration responsible for HF syndrome. To stop the HF epidemic will ultimately require protocols to reduce the progressive cardiomyocyte (CM) loss and to foster their regeneration. It is now generally accepted that mammalian CMs renew throughout life. However, this endogenous regenerative reservoir is insufficient to repair the extensive damage produced by AMI/IHD while the source and degree of CM turnover remains strongly disputed. Independent groups have convincingly shown that the adult myocardium harbors bona-fide tissue specific cardiac stem cells (CSCs). Unfortunately, recent reports have challenged the identity and the endogenous myogenic capacity of the c-kit expressing CSCs. This has hampered progress and unless this conflict is settled, clinical tests of repair/regenerative protocols are unlikely to provide convincing answers about their clinical potential. Here we review recent data that have eventually clarified the specific phenotypic identity of true multipotent CSCs. These cells when coaxed by embryonic cardiac morphogens undergo a precisely orchestrated myogenic commitment process robustly generating bona-fide functional cardiomyocytes. These data should set the path for the revival of further investigation untangling the regenerative biology of adult CSCs to harness their potential for HF prevention and treatment.     

How <Emphasis Type="Italic">Apis mellifera</Emphasis> Behaves with its Invasive Hornet Predator <Emphasis Type="Italic">Vespa velutina</Emphasis>?

Invasive species are now recognized as a major cause of native biodiversity loss worldwide. In the current deleterious context for pollinators, the invasive yellow-legged hornet, Vespa velutina, represents an additional threat to the domestic honeybee, Apis mellifera, in Europe. Therefore, understanding the impact of this predator on honeybee colonies is of major importance. In the present study, we tried to assess the impact of V. velutina on the honeybee foraging and defence behaviour based on the video monitoring of two hives. Balling behaviour is reported here for the first time under natural conditions in A. mellifera against V. velutina in Europe. Although these results are preliminary and should be carefully considered, we found that the number of hornets impacted honeybee foraging and defence behaviours. More defensive behaviours were notified in the hive, which survives slightly longer. This may suggest that selecting for more defensive colonies may provide an interesting perspective.     

Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress,serum deprivation or hypoxia mimetic CoCl2

Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5′ untranslated region (5′ UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5′ UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl₂, up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5′ UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed.     

Gut regulatory peptides in some Antarctic notothenioids

The presence and the comparative distribution of regulatory peptides and serotonin in the gut of four species of Antarctic notothenioid fishes [Cryodraco antarcticus and Chionodraco hamatus (Channichthyidae), and Trematomus bernacchii and Trematomus newnesi (Nototheniidae)], were immunohistochemically studied. In these species, numerous immunoreactive (IR) endocrine cells and nerve elements were detected. In the nototheniids most of the IR nerve fibres were of the intrinsic type, while most of the IR nerve fibres of the channichthyid intestine, besides insulin-like IR fibres, seemed to be of the extrinsic type. The intensity and frequency of immunopositivity are not the same in channnichthyids and nototheniids; the species belonging to the first family show many differences from the teleosts living in temperate water. The finding of insulin-like endocrine cells and nerve fibres in the gut wall of Cryodraco antarcticus is exceptional for vertebrates and deserves special attention.A preliminary account of this work was presented to the VII International Ichthyology Congress, Den Haag, 26–30 August 1991     

Mitochondrial Proliferation and Paradoxical Membrane Depolarization during Terminal Differentiation and Apoptosis in a Human Colon Carcinoma Cell Line

Herbimycin A, a tyrosine kinase inhibitor, induces cellular differentiation and delayed apoptosis in Colo-205 cells, a poorly differentiated human colon carcinoma cell line. Cell cycle analysis in conjunction with end labeling of DNA fragments revealed that G₂ arrest preceded apoptotic cell death. Ultrastructural examination of herbimycin-treated cells demonstrated morphologic features of epithelial differentiation, including formation of a microvillar apical membrane and lateral desmosome adhesions. A marked accumulation of mitochondria was also observed. Fluorometric analysis using the mitochondrial probes nonyl-acridine orange and JC-1 confirmed a progressive increase in mitochondrial mass. However these cells also demonstrated a progressive decline in unit mitochondrial transmembrane potential (ΔΨ_m) as determined by the ΔΨ_m-sensitive fluorescent probes rhodamine 123 and JC-1 analyzed for red fluorescence. In concert with these mitochondrial changes, Colo-205 cells treated with herbimycin A produced increased levels of reactive oxygen species as evidenced by oxidation of both dichlorodihydrofluorescein diacetate and dihydroethidium. Cell-free assays for apoptosis using rat-liver nuclei and extracts of Colo-205 cells at 24 h showed that apoptotic activity of Colo-205 lysates requires the early action of mitochondria. Morphological and functional mitochondrial changes were observed at early time points, preceding cleavage of poly (ADP-ribose) polymerase.

These results suggest that apoptosis in differentiated Colo-205 cells involves unrestrained mitochondrial proliferation and progressive membrane dysfunction, a novel mechanism in apoptosis.

     

Antagonism of Convulsions But Failure to Enhance GABAA Receptor Function by Felbamate in Mice Tolerant to Diazepam

The transfer of tolerance between drugs may indicate a common mode of action. The development of cross-tolerance to the anticonvulsant effect of felbamate after long-term treatment of mice with diazepam, a positive modulator of -aminobutyric acid (GABA)-mediated transmission, was therefore studied in order to clarify the mechanism of this action of felbamate. A challenge injection of felbamate, administered 36 h after the last dose of chronic diazepam treatment, antagonized convulsions elicited by administration of isoniazid. In contrast, felbamate had no effect on the isoniazid-induced increase in t-[³⁵S]butylbicyclophosphorothionate binding to cerebral cortical membranes of diazepam-tolerant mice. These results suggest that the action of felbamate on GA-BAergic transmission is not required for the anticonvulsant effect of this drug. This conclusion is consistent with studies that have indicated that the antiepileptic activity of felbamate depends on its modulatory activity at excitatory amino acid receptors.     

Tumour‐associated macrophages correlate with microvascular bed extension in colorectal cancer patients

Tumour‐associated macrophages (TAMs) represent pivotal components of tumour microenvironment promoting angiogenesis, tumour progression and invasion. In colorectal cancer (CRC), there are no conclusive data about the role of TAMs in angiogenesis‐mediated tumour progression. In this study, we aimed to evaluate a correlation between TAMs, TAM immunostained area (TAMIA) microvascular density (MVD), endothelial area (EA) and cancer cells positive to VEGF‐A (CCP‐VEGF‐A) in primary tumour tissue of locally advanced CRC patients undergone to radical surgery. A series of 76 patients with CRC were selected and evaluated by immunohistochemistry and image analysis. An anti‐CD68 antibody was employed to assess TAMs and TAMIA expression, an anti‐CD34 antibody was utilized to detect MVD and EA expression, whereas an anti‐VEGF‐A antibody was used to detect CCP‐VEGF‐A; then, tumour sections were evaluated by image analysis methods. The mean ± S.D. of TAMs, MVD and CCP‐VEGF‐A was 65.58 ± 21.14, 28.53 ± 7.75 and 63% ± 37%, respectively; the mean ± S.D. of TAMIA and EA was 438.37 ± 124.14μ² and 186.73 ± 67.22μ², respectively. A significant correlation was found between TAMs, TAMIA, MVD and EA each other (r ranging from 0.69 to 0.84; P ranging from 0.000 to 0.004). The high level of expression of TAMs and TAMIA in tumour tissue and the significant correlation with both MVD and EA illustrate that TAMs could represent a marker that plays an important role in promoting angiogenesis‐mediated CRC. In this context, novel agents killing TAMs might be evaluated in clinical trials as a new anti‐angiogenic approach.     

Chemical-Induced Read-Through at Premature Termination Codons Determined by a Rapid Dual-Fluorescence System Based on S. cerevisiae

Nonsense mutations generate in-frame stop codons in mRNA leading to a premature arrest of translation. Functional consequences of premature termination codons (PTCs) include the synthesis of truncated proteins with loss of protein function causing severe inherited or acquired diseases. A therapeutic approach has been recently developed that is based on the use of chemical agents with the ability to suppress PTCs (read-through) restoring the synthesis of a functional full-length protein. Research interest for compounds able to induce read-through requires an efficient high throughput large scale screening system. We present a rapid, sensitive and quantitative method based on a dual-fluorescence reporter expressed in the yeast Saccharomyces cerevisiae to monitor and quantitate read-through at PTCs. We have shown that our novel system works equally well in detecting read-through at all three PTCs UGA, UAG and UAA.     

Foot-shock stress enhances the increase of [35S]TBPS binding in the rat cerebral cortex and the convulsions induced by isoniazid

We report earlier that isoniazid and foot-shock stress individually increase the maximal number of [³⁵S]TBPS binding sites (B_max) measured ex vivo in unwashed membranes from rat cerebral cortex and that the increase due to both treatments are prevented by pretreatment in vivo with diazepam which alone induced a significant decrease in the total number of [³⁵S]TBPS binding sites. In the present paper, the effect of stress was studied on both the increase in [³⁵S]TBPS binding and the convulsant activity induced by isoniazid in unstressed rats. Isoniazid induced a time dependent increase in [³⁵S]TBPS binding. The isoniazid-induced increase in [³⁵S]TBPS binding was markedly potentiated by foot-shock stress. Moreover, foot-shock stress markedly reduced the latency to the appearance of generalized seizures induced by isoniazid (300 mg/kg s.c.). The results provide evidence that the in vivo inhibition of GABAergic transmission elicited by isoniazid results in an increase of [³⁵S]TBPS binding in the rats cerebral cortex. The finding that stress, like isoniazid, enhances [³⁵S]TBPS binding suggests that this treatment also inhibits the function of GABAergic synapses.