Experimental Evidence for a Revision in the Annotation of Putative Pyridoxamine 5'-Phosphate Oxidases P(N/M)P from Fungi

Pyridoxinamine 5''-phosphate oxidases (P(N/M)P oxidases) that bind flavin mononucleotide (FMN) and oxidize pyridoxine 5''-phosphate or pyridoxamine 5''-phosphate to form pyridoxal 5''-phosphate (PLP) are an important class of enzymes that play a central role in cell metabolism. Failure to generate an adequate supply of PLP is very detrimental to most organisms and is often clinically manifested as a neurological disorder in mammals. In this study, we analyzed the function of YLR456W and YPR172W, two homologous genes of unknown function from S. cerevisiae that have been annotated as putative P(N/M)P oxidases based on sequence homology. Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN. On the other hand, our analysis confirmed the enzymatic activity of Pdx3, the S. cerevisiae protein previously implicated in PLP biosynthesis by genetic and structural characterization. After a careful sequence analysis comparing the putative and confirmed P(N/M)P oxidases, we found that the protein domain (PF01243) that led to the YLR456W and YPR172W annotation is a poor indicator of P(N/M)P oxidase activity. We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function. This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases.     

Brazilian Red Propolis Attenuates Hypertension and Renal Damage in 5/6 Renal Ablation Model

The pathogenic role of inflammation and oxidative stress in chronic kidney disease (CKD) is well known. Anti-inflammatories and antioxidant drugs has demonstrated significant renoprotection in experimental nephropathies. Moreover, the inclusion of natural antioxidants derived from food and herbal extracts (such as polyphenols, curcumin and lycopene) as an adjuvant therapy for slowing CKD progression has been largely tested. Brazilian propolis is a honeybee product, whose anti-inflammatory, antimicrobial and antioxidant effects have been widely shown in models of sepsis, cancer, skin irritation and liver fibrosis. Furthermore, previous studies demonstrated that this compound promotes vasodilation and reduces hypertension. However, potential renoprotective effects of propolis in CKD have never been investigated. The aim of this study was to evaluate the effects of a subtype of Brazilian propolis, the Red Propolis (RP), in the 5/6 renal ablation model (Nx). Adult male Wistar rats underwent Nx and were divided into untreated (Nx) and RP-treated (Nx+RP) groups, after 30 days of surgery; when rats already exhibited marked hypertension and proteinuria. Animals were observed for 90 days from the surgery day, when Nx+RP group showed significant reduction of hypertension, proteinuria, serum creatinine retention, glomerulosclerosis, renal macrophage infiltration and oxidative stress, compared to age-matched untreated Nx rats, which worsened progressively over time. In conclusion, RP treatment attenuated hypertension and structural renal damage in Nx model. Reduction of renal inflammation and oxidative stress could be a plausible mechanism to explain this renoprotection.     

60 Hz Electric Field Changes the Membrane Potential During Burst Phase in Pancreatic β-Cells: In Silico Analysis

The production, distribution and use of electricity can generate low frequency electric and magnetic fields (50–60 Hz). Considering that some studies showed adverse effects on pancreatic β-cells exposed to these fields; the present study aimed to analyze the effects of 60 Hz electric fields on membrane potential during the silent and burst phases in pancreatic β-cells using a mathematical model. Sinusoidal 60 Hz electric fields with amplitude ranging from 0.5 to 4 mV were applied on pancreatic β-cells model. The sinusoidal electric field changed burst duration, inter-burst intervals (silent phase) and spike sizes. The parameters above presented dose-dependent response with the voltage amplitude applied. In conclusion, theoretical analyses showed that a 60 Hz electric field with low amplitudes changes the membrane potential in pancreatic β-cells.     

Can the structure of a riparian forest remnant influence stream water quality? A tropical case study

In rural areas, riparian forest remnants can be very important for the maintenance and improvement of stream water quality. We evaluated if a forest remnant influenced stream water quality, and if these results were influenced by forest structure. We sampled reaches from 140 m upstream of the remnant edge until 600 m within the forest remnant. Electric conductivity (EC) and ammonium concentrations decreased as the stream flowed through the remnant, whereas dissolved oxygen, total phosphorus (P), total dissolved P, organic P, and silicate concentrations increased along the remnant. Variation in forest structure was due to a gradient in forest stratification versus tree height and diameter at breast height, and a gradient in tree density versus basal area. More stratified parts of the forest, with smaller trees, resulted in lower EC values and concentrations of total nitrogen and nitrite, whereas higher density of trees resulted in lower levels of total and dissolved P, creating heterogeneity at very local scales. The overall mean influence of this riparian forest remnant improved stream water quality, suggesting that forest remnants have local effects that can be important when managing stream water quality at larger spatial scales.     

Experimental Evidence that Phenylalanine Provokes Oxidative Stress in Hippocampus and Cerebral Cortex of Developing Rats

High levels of phenylalanine (Phe) are the biochemical hallmark of phenylketonuria (PKU), a neurometabolic disorder clinically characterized by severe mental retardation and other brain abnormalities, including cortical atrophy and microcephaly. Considering that the pathomechanisms leading to brain damage and particularly the marked cognitive impairment in this disease are poorly understood, in the present study we investigated the in vitro effect of Phe, at similar concentrations as to those found in brain of PKU patients, on important parameters of oxidative stress in the hippocampus and cerebral cortex of developing rats. We found that Phe induced in vitro lipid peroxidation (increase of TBA-RS values) and protein oxidative damage (sulfhydryl oxidation) in both cerebral structures. Furthermore, these effects were probably mediated by reactive oxygen species, since the lipid oxidative damage was totally prevented by the free radical scavengers α-tocopherol and melatonin, but not by L-NAME, a potent inhibitor of nitric oxide synthase. Accordingly, Phe did not induce nitric oxide synthesis, but significantly decreased the levels of reduced glutathione (GSH), the major brain antioxidant defense, in hippocampus and cerebral cortex supernatants. Phe also reduced the thiol groups of a commercial GSH solution in a cell-free medium. We also found that the major metabolites of Phe catabolism, phenylpyruvate, phenyllactate and phenylacetate also increased TBA-RS levels in cerebral cortex, but to a lesser degree. The data indicate that Phe elicits oxidative stress in the hippocampus, a structure mainly involved with learning/memory, and also in the cerebral cortex, which is severely damaged in PKU patients. It is therefore presumed that this pathomechanism may be involved at least in part in the severe cognitive deficit and in the characteristic cortical atrophy associated with dysmyelination and leukodystrophy observed in this disorder.     

Seroepidemiological Survey of Paracoccidioidomycosis Infection Among Urban and Rural Dogs from Uberaba,Minas Gerais,Brazil

There is some evidence that dogs can be naturally infected by Paracoccidioides brasiliensis in endemic areas of paracoccidioidomycosis. In order to evaluate canine infection with this fungus, a survey with 149 urban and 126 rural dogs was carried out using ELISA and intradermal tests with the gp43 antigen of P. brasiliensis in Uberaba, Minas Gerais state of Brazil. Forty-one out of 149 urban dogs were euthanatized and had their lungs, liver and spleen removed. One slice from each viscera was processed for histopathological examination and the remaining was homogenized and then cultivated on mycobiotic agar at room temperature and Fava-Netto medium at 35°C and observed for 12 weeks. Of urban dogs, 75 (50.3%) were small adult females, 56 (36%) were strays, while 93 (64%) had been donated to the municipal zoonosis control center. Nine (6.2%) had a positive intradermal test without statistical differences regarding gender, race, nutritional status or origin. No colonies with microscopic or morphology appearances resembling P. brasiliensis were isolated, nor granulomatous process or fungal structures were observed from histopathological examination. Eighty (53.6%) of the urban dogs presented seroreactivity, without statistical differences regarding gender, race, nutritional state, origin, or positive intradermal test. Of 126 rural dogs, 102 (80.5%) presented antibodies against gp43 antigen, and this was statistically significant in relation to the reactivity detected in urban dogs (P = 0.0001). Thus, dogs are commonly infected with P. brasiliensis, but they probably present natural resistance to develop paracoccidioidomycosis.     

Characteristics of Environmental <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.     

A laccase exclusively expressed by Metarhizium anisopliae during isotropic growth is involved in pigmentation,tolerance to abiotic stresses and virulence

Insect pathogenic fungi including Metarhizium anisopliae offer an environmentally friendly alternative to chemical pesticides. However, their use has been limited by their relatively slow killing speed compared to chemicals and low tolerance to abiotic stresses. We report here on a class 1 laccase (MLAC1) that is involved in both virulence and tolerance to environmental stresses. Mlac1 is expressed during isotropic growth (swelling) but not during polarized growth (e.g., germ tubes and hyphae); Mlac1 is therefore expressed exclusively in the later stages of conidiation and in blastospores when M. anisopliae is living as a saprophyte. During infection processes, Mlac1 is also expressed by appressoria (infection structures) on the cuticle surface and hyphal bodies inside the insect haemocoel. Disrupting Mlac1 reduced virulence to caterpillars because of impaired appressoria and delayed post-infection events. It also produced a yellow-conidia phenotype with increased conidial susceptibility to heat shock (45 °C for 2 h) and UV-B stress. The relationship between M. anisopliae’s pigment-synthesis pathway and its adaptation to diverse natural habitats is discussed.     

Identification of a new hexadentate iron chelator capable of restricting the intramacrophagic growth of Mycobacterium avium

Iron accumulation has been suggested to contribute to an increase of the susceptibility to mycobacterial infections. In this study we tested the effect of an array of iron chelating ligands of the 3-hydroxy-4-pyridinone family, in the intramacrophagic growth of Mycobacterium avium. We found that bidentate chelators, namely N-alkyl-3-hydroxy-4-pyridinones and N-aryl-3-hydroxy-4-pyridinones, did not affect the growth of M. avium inside mouse macrophages. In the case of the hexadentate chelators, those synthesized using an alkylamine (CP262) or a benzene ring (CP252) to link the three bidentate units, did not have an inhibitory effect on intramacrophagic growth of M. avium while those synthesized from a tripodal structure to anchor the bidentate units were capable of inhibiting the intramacrophagic growth of M. avium. The molecule we designated CP777 had the strongest inhibitory activity. The growth-reducing activity of CP777 was abrogated when this molecule was saturated with iron. These results confirm that iron deprivation, by the use of iron chelating compounds, restricts M. avium growth and that new iron chelators offer an approach to controlling mycobacterial infections.     

Analysis of apoptosis and Bcl-2 expression in polar forms of leprosy

Apoptosis eliminates pathogen-infected cells. Its modulation can influence the course of infections, permitting the survival of intracellular pathogens. In leprosy, which presents several clinical manifestations related to bacillary burden and host immune status, the mechanisms responsible for the persistence of the bacillus are unknown. Few studies have focused on apoptosis over the disease spectrum and as a defense mechanism against Mycobacterium leprae. We evaluated apoptosis using terminal transferase dUTP nick end labeling and the expression of Bcl-2 by immunohistochemistry in skin lesions from 11 tuberculoid and 15 lepromatous leprosy patients. Each specimen was evaluated by determining the number of positive cells in 10 fields at × 400 magnification. We observed a higher number of apoptotic cells in tuberculoid lesions in comparison with lepromatous leprosy (42.5 cells per 10 fields vs. 11.5 cells per 10 fields, P<0.0001). Expression of Bcl-2, conversely, was larger in lepromatous than in tuberculoid samples (172.0 cells per 10 fields vs. 17.7 cells per 10 fields, P<0.0001). These observations suggest modulation of apoptosis in leprosy, primarily in lepromatous patients, for which the decrease in cell death could support M. leprae survival and contribute to the success of infection. Conversely, in tuberculoid patients, apoptosis could contribute to reducing propagation of the bacillus.