Genotoxicity of Nicotiana tabacum leaves on Helix aspersa

Tobacco farmers are routinely exposed to complex mixtures of inorganic and organic chemicals present in tobacco leaves. In this study, we examined the genotoxicity of tobacco leaves in the snail Helix aspersa as a measure of the risk to human health. DNA damage was evaluated using the micronucleus test and the Comet assay and the concentration of cytochrome P450 enzymes was estimated. Two groups of snails were studied: one fed on tobacco leaves and one fed on lettuce (Lactuca sativa L) leaves (control group). All of the snails received leaves (tobacco and lettuce leaves were the only food provided) and water ad libitum. Hemolymph cells were collected after 0, 24, 48 and 72 h. The Comet assay and micronucleus test showed that exposure to tobacco leaves for different periods of time caused significant DNA damage. Inhibition of cytochrome P450 enzymes occurred only in the tobacco group. Chemical analysis indicated the presence of the alkaloid nicotine, coumarins, saponins, flavonoids and various metals. These results show that tobacco leaves are genotoxic in H. aspersa and inhibit cytochrome P450 activity, probably through the action of the complex chemical mixture present in the plant.     

Laminin‐γ1 chain and stress inducible protein 1 synergistically mediate PrPC‐dependent axonal growth via Ca2+ mobilization in dorsal root ganglia neurons

Prion protein (PrP^C) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP^C interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP^C co‐opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross‐talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP^C‐mediated axonogenesis in peripheral neurons in response to STI1 and laminin‐γ1 chain‐derived peptide (Ln‐γ1). STI1 and Ln‐γ1 promoted robust axonogenesis in wild‐type neurons, whereas no effect was observed in neurons from PrP^C‐null mice. PrP^C binding to Ln‐γ1 or STI1 led to an increase in intracellular Ca²⁺ levels via distinct mechanisms: STI1 promoted extracellular Ca²⁺ influx, and Ln‐γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln‐γ1, but depends on, C‐type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP^C‐mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP^C. These results suggest a role for PrP^C as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.     

Distribution of Chitinases in the Entomopathogen <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine in Protein Secretion

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.     

Regulation of extracellular chitinases and proteases in the entomopathogen and acaricide Metarhizium anisopliae

Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS-PAGE.     

20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated

The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.     

Expression and characterization of the 42 kDa chitinase of the biocontrol fungus Metarhizium anisopliae in Escherichia coli

Albeit Metarhizium anisopliae is the best-characterized entomopathogenic fungus, the role of some hydrolytic enzymes during host cuticle penetration has not yet been established. Three chitinase genes (chit1, chi2, chi3) from Metarhizium have already been isolated. To characterize the chitinase coded by the chit1 gene, we expressed the active protein (CHIT42) in Escherichia coli using a T7-based promoter expression vector. The recombinant protein, CHIT42, is active against glycol chitin and synthetic N-acetylglucosamine (GlcNAc) dimer and tetramer substrates. These activities suggest that the recombinant CHIT42 acts as an endochitinase.     

Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis

The direct agglutination test (DAT) based on a freeze-dried antigen and the rK39 dipstick test were evaluated for the sero-diagnosis of visceral leishmaniasis (VL). The sensitivity and specificity of both tests were determined using sera from confirmed VL patients (n = 21), healthy controls (n = 19) and from patients with other confirmed infectious diseases (n = 42). The DAT had a sensitivity and a specificity of 100%. The rK39 had a sensitivity of 85.7% and a specificity of 82%. Both tests were also used to screen blood samples of confirmed VL patients (n = 15) and serum samples of VL suspects (n = 61). The DAT found all blood samples of confirmed VL patients positive and tested 98.4% of the serum samples of the VL suspects positive. In contrast, rK39 detected in 9/15 blood samples (60%) antibodies against Leishmania chagasi and found 85.3% of the serum samples of the suspected patients positive. Although the rK39 dipstick is more rapid and user friendlier than the DAT, the latter has a superior sensitivity and specificity. Furthermore, the reagents used for DAT do not require cold storage, whereas the buffer of the rK39 must be stored at 4oC. Therefore, the DAT is the most suitable test for the sero-diagnosis of VL under field conditions.     

Differential regulation of prostaglandin E2 and thromboxane A2 production in human monocytes: implications for the use of cyclooxygenase inhibitors

There is an autocrine relationship between eicosanoid and cytokine synthesis, with the ratio of prostaglandin E2 (PGE2)/thromboxane A2 (TXA2) being one of the determinants of the level of cytokine synthesis. In monocytes, cyclooxygenase type 1 (COX-1) activity appears to favor TXA2 production and COX-2 activity appears to favor PGE2 production. This has led to speculation regarding possible linkage of COX isozymes with PGE and TXA synthase. We have studied the kinetics of PGE2 and TXA2 synthesis under conditions that rely on COX-1 or -2 activity. With small amounts of endogenously generated prostaglandin H2 (PGH2), TXA2 synthesis was greater than PGE2. With greater amounts of endogenously generated PGH2, PGE2 synthesis was greater than TXA2. Also, TXA synthase was saturated at lower substrate concentrations than PGE synthase. This pattern was observed irrespective of whether PGH2 was produced by COX-1 or COX-2 or whether it was added directly. Furthermore, the inhibition of eicosanoid production by the action of nonsteroidal anti-inflammatory drugs or by the prevention of COX-2 induction with the p38 mitogen-activated protein kinase inhibitor SKF86002 was greater for PGE2 than for TXA2. It is proposed that different kinetics of PGE synthase and TXA synthase account for the patterns of production of these eicosanoids in monocytes under a variety of experimental conditions. These properties provide an alternative explanation to notional linkage or compartmentalization of COX-1 or -2 with the respective terminal synthases and that therapeutically induced changes in eicosanoid ratios toward predominance of TXA2 may have unwanted effects in long-term anti-inflammatory and anti-arthritic therapy.     

Cellular senescence and autophagy of myoepithelial cells are involved in the progression of in situ areas of carcinoma ex-pleomorphic adenoma to invasive carcinoma. An in vitro model

During tumor invasion, benign myoepithelial cells of carcinoma ex-pleomorphic adenoma (CXPA) surround malignant epithelial cells and disappear. The mechanisms involved in the death and disappearance of these myoepithelial cells were investigated via analysis of the expression of regulatory proteins for apoptosis, autophagy and cellular senescence in an in situ in vitro model. Protein expression relating to apoptosis (Bax, Bcl-2, Survivin), autophagy (Beclin-1, LC3B) and cellular senescence (p21, p16) was evaluated using indirect immunofluorescence. β-galactosidase expression was assessed via histochemistry. Biopsies of CXPA (ex vivo) allowed immunhistochemical evaluation of p21 and p16, whilst LC3B, p21 and p16 protein expression was analyzed by western blotting. In the in vitro model, the myoepithelial cells were positive for LC3B (cytoplasm) and p21 (nucleus), whilst in vivo positivity for p21 and p16 was observed. In vitro, β-galactosidase activity increased in the myoepithelial cells over time. Western blotting analysis revealed an increased LC3B, p16 and p21 expression in the myoepithelial cells with previous contact with the malignant cells when compared with those without contact. The investigation of behavior of benign myoepithelial cells in ductal areas of CXAP revealed that the myoepithelial cells are involved in the autophagy-senescence phenotype that subsequently leads to their disappearance.     

Rotavirus and adenovirus in Rondônia

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viral gastroenteritis is a global problem in infants and young children. In this study the incidence of diarrhea was assessed in 877 hospitalized children under five years old, over a period of 24 months and distributed in 470 cases of diarrhea and 407 age-matched group with other pathologies, as control group. Two antigen detection techniques based on enzyme immunoassay (EIA) and latex particles were used for detection of rotavirus and adenovirus. Rotavirus A was a major cause of gastroenteritis with 23.6% of cases, being 90% of these cases in young children. Adenovirus infections was detected by EIA with frequency of 6.4%. Rotavirus and adenovirus were detected in 10.1 and 1.7% of stools from control group, respectively. Interestingly, the frequency of the youngest children in the control group excreting Rotavirus A was comparable to that detected in stools from diarrheic children. We cannot rule out the existence of other enteric viruses because the etiology of 171 cases of diarrhea was not determined and active search for astrovirus and calicivirus was not done. This is the first study that shows the presence of enteric viruses in the infantile population from Western Brazilian Amazonia and it was important to help physicians in the treatment of viral gastroenteritis.