Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis

The objective of this work is to investigate the utilization of two abundant agricultural residues in Brazil for the production and application of cellulolytic enzymes. Different materials obtained after pretreatment of sugarcane bagasse, as well as pure synthetic substrates, were considered for cellulase production by Penicillium funiculosum. The best results for FPase (354 U L^?1) and β-glucosidase (1,835 U L^?1) production were observed when sugarcane bagasse partially delignified cellulignin (PDC) was used. The crude extract obtained from PDC fermentation was then partially characterized. Optimal temperatures for cellulase action ranged from 52 to 58°C and pH values of around 4.9 contributed to maximum enzyme activity. At 37°C, the cellulases were highly stable, losing less than 15% of their initial activity after 23 h of incubation. There was no detection of proteases in the P. funiculosum extract, but other hydrolases, such as endoxylanases, were identified (147 U L^?1). Finally, when compared to commercial preparations, the cellulolytic complex from P. funiculosum showed more well-balanced amounts of β-glucosidase, endo- and exoglucanase, resulting in the desired performance in the presence of a lignocellulosic material. Cellulases from this filamentous fungus had a higher glucose production rate (470 mg L^?1 h^?1) when incubated with corn cob than with Celluclast^®, GC 220^® and Spezyme^® (312, 454 and 400 mg L^?1 h^?1, respectively).     

The cross-road between the mechanisms of protein folding and aggregation; study of human stefin B and its H75W mutant

The role of the aromatic residue at site 75 to protein stability, the mechanism of folding and the mechanism of amyloid-fibril formation were investigated for the human stefin B variant (bearing Y at site 31) and its point mutation H75W. With an aim to reveal the conformation at the cross-road between folding and aggregation, first, the kinetics of folding and oligomer formation by human stefin B(Y31) variant were studied. It was found to fold in three kinetic phases at pH 4.8 and 10% TFE; the pH and solvent conditions that transform the protein into amyloid fibrils at longer times. The same pH leads to the formation of native-like intermediate (known from previous studies of this variant), meaning that the process of folding and amyloid-fibril formation share the same structural intermediate, which is in this case native-like and dimeric. At pH 5.8 and 7.0 stefin B folded to the native state in four kinetic phases over two intermediates. In distinction, the mutant H75W did not fold to completion, ending in intermediate states at all pH values studied: 4.8, 5.8 and 7.0. At pH 4.8 and 5.8, the mutant folded in one kinetic phase to the intermediate of the “molten globule” type, which leads to the conclusion that its mechanism of folding differs from the one of the parent stefin B at the same pH. At pH 7.0 the mutant H75W folded in three kinetic phases to a native-like intermediate, analogous to folding of stefin B at pH 4.8.     

Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1(+)/CD127(-) HCV-specific CD8(+) cells targeting the virus in chronic hepatitis C virus infection

PD-1 molecule promotes anergy and IL-7 receptor (CD127) induces an anti-apoptotic effect on T cells. Correlation between PD-1/CD127 phenotype and hepatitis C virus (HCV)-specific CD8⁺ cell reactivity in resolved infection (RI) after treatment and persistent HCV-infection (PI) was analysed. Directly ex vivo, PD-1 and CD127 expression on HCV-specific CD8⁺ cells displayed a positive and negative correlation, respectively with viraemia. Proliferation after stimulation on PD-1⁻/CD127⁺ cells from RI cases was preserved, while it was impaired on PD-1⁺/CD127⁻ cells from PI patients. PD1⁺/CD127⁺ population was observed in PI, and these maintained expansion ability but they did not target the virus. Frequency of PI cases with HCV-specific CD8⁺ cell proliferation increased after anti-PD-L1 and anti-apoptotic treatment. Bim expression on HCV-specific CD8⁺ cells from PI patients was enhanced. In conclusion, during chronic HCV infection non-reactive HCV-specific CD8⁺ cells targeting the virus are PD-1⁺/CD127⁻/Bim⁺ and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in vitro reactivity.     

Sexual selection drives the evolution of antiaphrodisiac pheromones in butterflies

Competition for mates has resulted in sophisticated mechanisms of male control over female reproduction. Antiaphrodisiacs are pheromones transferred from males to females during mating that reduce attractiveness of females to subsequent courting males. Antiaphrodisiacs generally help unreceptive females reduce male harassment. However, lack of control over pheromone release by females and male control over the amount transferred provides males an opportunity to use antiaphrodisiacs to delay remating by females that have returned to a receptive state. We propose a model for the evolution of antiaphrodisiacs under the influence of intrasexual selection, and determine whether changes in this signal in 11 species of Heliconius butterflies are consistent with two predictions of the model. First, we find that as predicted, male-contributed chemical mixtures are complex and highly variable across species, with limited phylogenetic signal. Second, differences in rates of evolution in pheromone composition between two major clades of Heliconius are as expected: the clade with a greater potential for male-male competition (polyandrous) shows a faster rate of divergence than the one with typically monoandrous mating system. Taken together, our results provide evidence that for females, antiaphrodisiacs can be both honest signals of receptivity (helping reduce harassment) and chastity belts (a male-imposed reduction in remating).     

Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA

Trans-cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg(2+) concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg(2+). Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons.     

Prevalence of known mutations in the MEFV gene in a population screening with high rate of carriers

The Familial Mediterranean Fever (FMF) shows an autosomal recessive pattern of inheritance and affects certain ethnic groups. Disease is caused by mutations in MEFV gene and more than 180 mutations have been defined in affected individuals. Current study aimed to determine the frequency-type of the mutations for MEFV gene in Sivas??middle Anatolian city. The cohort was composed of 3340 patients. MEFV gene mutations were studied by multiplex PCR based reverse hybridization stripAssay method. Patients?? clinical features were; family history: 68%, erysipelas-like erythema: 17.6%, fever: 89.9%, abdominal pain: 84.2%, peritonitis: 90.2%, arthritis: 33%, pleuritis: 14.2%, parental consanguinity: 21.2%. Current results revealed that M694V is the most frequent mutation (43.12%), followed by E148Q (20.18), M680I(G/C) (15.00%) and V726A (11.32%). The study population has a high rate of carriers and the E148Q mutation frequency was found to be highest when compared to the other regions of Turkey and other Mediterranean groups.     

Use of in vivo and in vitro systems to select Leishmania amazonensis expressing green fluorescent protein

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.     

Hepatitis B and C in the hemodialysis unit of Tocantins,Brazil: serological and molecular profiles

A survey was conducted in the hemodialysis population of the state of Tocantins, Brazil, aiming to assess the prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, to analyze associated risk factors, and also to investigate these viruses genotypes distribution. During January and March 2001, all patients (n = 100) were interviewed at the unique dialysis unit in Tocantins. Blood samples were collected and serum samples were screened for HBV serological markers. Hepatitis B surface antigen positive samples were tested for HBV DNA. All samples were also tested for anti-HCV antibodies and HCV RNA. An overall prevalence of 45% was found for HBV infection (4% were HBsAg/anti-HBc positive, 2% were anti-HBc only and 39% had anti-HBc/anti-HBs markers). Concerning HCV infection, anti-HCV and HCV RNA were detected in 13% and 14% of the subjects, respectively. Three patients were HCV RNA positive and anti-HCV negative, resulting in an overall HCV prevalence of 16%. Univariate analysis of risk factors showed that only shift and length of tile on hemodialysis were associated with HBV and HCV positivity respectively. Among the four HBsAg-positive samples, HBV DNA was detected in three of them, which were identified as genotype A by restriction fragment length polymorphism (RFLP) analysis. All 14HCV RNA-positive samples were genotyped by INNO-LiPA. Genotypes la and 3a were found in 85% and 15%, respectively. The present data show low HBsAg and HCV prevalence rates. The risk factors associated with HBV and HCV positivity suggest that nosocomial transmission may influence in spreading these viruses in the dialysis unit studied.