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1.
Clin Exp Immunol ; 171(2): 210-9, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23286948

ABSTRACT

Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis (MDR-TB) and non-tuberculous mycobacteria (NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB (NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4(+) CD25(+-) forkhead box protein 3 (FoxP3)(+) and CD4(+) CD25(+) CD127(-) regulatory T (T(reg) ) cells when compared to NR-TB. Increased numbers of T(reg) cells and interleukin (IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor (TGF)-ß was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target (ESAT)-6, but not purified protein derivative (PPD), showed a lower frequency of CD4(+) /interferon (IFN)-γ(+) T cells and enhanced CD4(+) CD25(+) FoxP3(+) , CD4(+) CD25(+) CD127(-) and CD4(+) CD25(+) IL-10(+) T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL-10Rα decreased the CD4(+) CD25(+) FoxP3(+) frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4(+) CD25(+) T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for T(reg) /T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms.


Subject(s)
Immune Tolerance , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Tuberculosis, Multidrug-Resistant/immunology , Tuberculosis, Pulmonary/immunology , Adult , Aged , Antigens, Bacterial/immunology , Antigens, CD/metabolism , Bacterial Proteins/immunology , Cells, Cultured , Cytokines/immunology , Female , Forkhead Transcription Factors/metabolism , Humans , Immunophenotyping , Isoniazid/therapeutic use , Male , Middle Aged , Rifampin/therapeutic use , T-Lymphocyte Subsets/drug effects , T-Lymphocytes, Regulatory/drug effects , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/microbiology , Young Adult
2.
Lett Appl Microbiol ; 56(4): 283-90, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23350659

ABSTRACT

The discharge of highly coloured synthetic dye effluents into rivers and lakes is harmful to the water bodies, and therefore, intensive researches have been focussed on the decolorization of wastewater by biological, physical or chemical treatments. In the present study, 12 basidiomycetes strains from the genus Pleurotus, Trametes, Lentinus, Peniophora, Pycnoporus, Rigidoporus, Hygrocybe and Psilocybe were evaluated for decolorization of the reactive dyes Cibacron Brilliant Blue H-GR and Cibacron Red FN-2BL, both in solid and liquid media. Among the evaluated fungi, seven showed great ability to decolorize the synthetic textile effluent, both in vivo (74-77%) or in vitro (60-74%), and laccase was the main ligninolytic enzyme involved on dyes decolorization. Pleurotus ostreatus, Trametes villosa and Peniophora cinerea reduced near to 60% of the effluent colour after only 1 h of treatment. The decolorization results were still improved by establishing the nitrogen source and amount to be used during the fungal strains cultivation in synthetic medium previous their action on the textile effluent, with yeast extract being a better nitrogen source than ammonium tartarate. These results contribute for the development of an effective microbiological process for decolorization of dye effluents with reduced time of treatment.


Subject(s)
Basidiomycota/metabolism , Coloring Agents/metabolism , Laccase/metabolism , Basidiomycota/enzymology , Basidiomycota/growth & development , Culture Media , Hydrogen-Ion Concentration , Nitrogen/metabolism , Oxidation-Reduction , Salts , Textiles , Wastewater
3.
World J Microbiol Biotechnol ; 28(1): 113-9, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22806786

ABSTRACT

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of α-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.


Subject(s)
Cellulase/biosynthesis , Fungal Proteins/biosynthesis , Glycoside Hydrolases/biosynthesis , Thermoascus/enzymology , Biomass , Biotechnology , Disaccharides/metabolism , Enzyme Induction , Hydrolysis , Kinetics , Thermoascus/growth & development , Thermoascus/metabolism , Xylans/metabolism
4.
Bioresour Technol ; 99(7): 2471-5, 2008 May.
Article in English | MEDLINE | ID: mdl-17583498

ABSTRACT

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to LiP of Phanerochaete chrysosporium and a total inhibition of LiP was observed.


Subject(s)
Peroxidases/isolation & purification , Shiitake Mushrooms/enzymology , Biomass , Molecular Weight , Peroxidases/biosynthesis , Peroxidases/chemistry , Substrate Specificity
5.
J Appl Microbiol ; 104(1): 185-93, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17850312

ABSTRACT

AIMS: The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability. METHODS AND RESULTS: W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe3+-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe3+-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible. CONCLUSIONS: W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.


Subject(s)
Fungal Proteins/metabolism , Industrial Microbiology , Iron/metabolism , Plant Diseases/microbiology , Polyporales/metabolism , Wood , Electrophoresis, Polyacrylamide Gel , FMN Reductase/metabolism , Iron/pharmacology , Iron Chelating Agents/metabolism , Mycology , Polyporales/growth & development , Silver Staining
6.
Appl Biochem Biotechnol ; 141(1): 37-50, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17625265

ABSTRACT

There are many changes, both qualitative and quantitative, in eucalypt waste during growth and fructification of Lentinula edodes. Wet chemical analysis and near-infrared (NIR) spectroscopy were used in conjunction with multivariate regression and principal components analysis to monitor biodegradation of eucalyptus waste during growth of several L. edodes strains. Weight and component losses of eucalypt residue after biodegradation by L. edodes strains were compared for periods of 1 to 5 mo. Decrease in cellulose, hemicellulose, and lignin contents occurred, however it was not concomitant. Measurement of lignin degradation by NIR and wet chemical analysis indicated its attack in the early stages of biodegradation. Selective lignin degradation by L. edodes was observed up to 2 mo of biodegradation for strains DEBIQ and FEB-14. One group of degraded substrate was identified based on the principal component analysis (PCA) of the data on their biodegradation time. Samples treated for 5 months by L. edodes strains (DEBIQ, UFV or FEB-14) differed from other, but no discrimination was observed among them. By the end of 5 mo, NIR analyses showed decrease of about 18-47% cellulose, 35-47% polyose and 39-60% lignin. These data were used for comparison with those obtained by wet chemical method for the degradation of the substrate by other five L. edodes strains cultivated at the same conditions. NIR calibration developed in this study was proven to be perfectly suitable as an analytical method to predict the changes in lignocellulose composition during biodegradation.


Subject(s)
Cellulose/metabolism , Eucalyptus/microbiology , Industrial Waste/prevention & control , Lignin/metabolism , Shiitake Mushrooms/isolation & purification , Shiitake Mushrooms/physiology , Spectroscopy, Near-Infrared/methods , Biodegradation, Environmental , Shiitake Mushrooms/classification , Species Specificity
7.
Water Sci Technol ; 55(6): 1-7, 2007.
Article in English | MEDLINE | ID: mdl-17486828

ABSTRACT

The aim of this work was to evaluate the effect of chelator mediated-Fenton reaction (CMFR) on the chemical oxygen demand (COD) removal from bleaching kraft mill effluent. Effluent treatments were carried out to study the effect of the chelator 3, 4-dihydroxyphenylacetic acid (DOPAC), Fe3+ and H2O2 concentrations on COD removal. For optimization of COD removal, the methodology of statistical experimental design was employed. The estimated second-order polynomial multiple regression model predicts a maximum COD removal of 75.5% (COD/COD0 = 0.245) at the confidence level of 95%. Analysis of variance (ANOVA) showed a good coefficient of determination (R2) value of 0.90, thus ensuring a satisfactory adjustment of the second-order regression model with the experimental data. Indeed, CMFR proved to be a potential process to treat industrial effluents characterized by its high COD.


Subject(s)
Free Radicals/metabolism , Research Design/statistics & numerical data , Waste Disposal, Fluid/methods , Water Pollutants, Chemical/metabolism , Water Pollution/prevention & control , 3,4-Dihydroxyphenylacetic Acid/pharmacology , Analysis of Variance , Chelating Agents/pharmacology , Dose-Response Relationship, Drug , Hydrogen Peroxide/pharmacology , Industrial Waste , Iron , Oxidants/pharmacology , Oxygen/chemistry , Oxygen/metabolism , Paper , Regression Analysis , Surface Properties
8.
Lett Appl Microbiol ; 44(1): 7-12, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17209807

ABSTRACT

AIMS: To investigate the in vitro production of metal-chelating compounds by ectomycorrhizal fungi collected from pine plantations in southern Chile. METHODS AND RESULTS: Scleroderma verrucosum, Suillus luteus and two isolates of Rhizopogon luteolus were grown in solid and liquid modified Melin-Norkans (MMN) media with and without iron addition and the production of iron-chelating compounds was determined by Chrome Azurol S (CAS) assay. The presence of hydroxamate and catecholate-type compounds and organic acids was also investigated in liquid medium. All isolates produced iron-chelating compounds as detected by CAS assay, and catecholates, hydroxamates as well as oxalic, citric and succinic acids were also detected in all fungal cultures. Scleroderma verrucosum produced the greatest amounts of catecholates and hydroxamates whereas the highest amounts of organic acids were detected in S. luteus. Nevertheless, the highest catecholate, hydroxamate and organic acid concentrations did not correlate with the highest CAS reaction which was observed in R. luteolus (Yum isolate). CONCLUSIONS: Ectomycorrhizal fungi produced a variety of metal-chelating compounds when grown in liquid MMN medium. However, the addition of iron to all fungi cultures reduced the CAS reaction, hydroxamate and organic acid concentrations. Catecholate production was affected differently by iron, depending on the fungal isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: The ectomycorrhizal fungi described in this study have never been reported to produce metal-chelating compound production. Moreover, apart from some wood-rotting fungi, this is the first evidence of the presence of catecholates in R. luteolus, S. luteus and S. verrucosum cultures.


Subject(s)
Basidiomycota/metabolism , Culture Media/chemistry , Pinus sylvestris/microbiology , Siderophores/biosynthesis , Chelating Agents/isolation & purification , Chelating Agents/pharmacology , Hydroxamic Acids/isolation & purification , Iron/metabolism , Microbiological Techniques , Siderophores/isolation & purification
9.
J Appl Microbiol ; 101(2): 480-6, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16882157

ABSTRACT

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.


Subject(s)
Basidiomycota/enzymology , Cellulose/metabolism , Environmental Microbiology , Glucose/biosynthesis , Wood , beta-Glucosidase/metabolism , Biodegradation, Environmental , Bioreactors , Cellulase/analysis , Cellulase/metabolism , Electrophoresis, Polyacrylamide Gel , Hot Temperature , Hydrogen-Ion Concentration , Pinus taeda , Substrate Specificity , beta-Glucosidase/analysis
10.
Braz J Med Biol Res ; 38(11): 1643-7, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16258633

ABSTRACT

To evaluate the human T-cell lymphotropic virus type I (HTLV-I) proviral DNA load among asymptomatic HTLV-I-infected carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), real time PCR using TaqMan probes for the pol gene was performed in two million peripheral blood mononuclear cells (PBMC). The albumin gene was the internal genomic control and MT2 cells were used as positive control. The results are reported as copies/10,000 PBMC, and the detection limit was 10 copies. A total of 89 subjects (44 HAM/TSP and 45 healthy HTLV-I-infected carriers) followed up at the Institute of Infectious Diseases "Emilio Ribas" and in the Neurology Division of Hospital of Clínicas were studied. The asymptomatic HTLV-I-infected carriers had a median number of 271 copies (ranging from 5 to 4756 copies), whereas the HAM/TSP cases presented a median of 679 copies (5-5360 copies) in 10,000 PBMC. Thus, HAM/TSP patients presented a significantly higher HTLV-I proviral DNA load than healthy HTLV-I carriers (P = 0.005, one-way Mann-Whitney test). As observed in other persistent infections, proviral DNA load quantification may be an important tool for monotoring HTLV-I-infected subjects. However, long-term follow-up is necessary to validate this assay in the clinical setting.


Subject(s)
DNA, Viral/analysis , Human T-lymphotropic virus 1/genetics , Paraparesis, Tropical Spastic/virology , Proviruses/genetics , Case-Control Studies , DNA, Viral/genetics , DNA, Viral/immunology , Human T-lymphotropic virus 1/immunology , Humans , Leukocytes, Mononuclear/immunology , Paraparesis, Tropical Spastic/immunology , Polymerase Chain Reaction , Proviruses/immunology , Viral Load
11.
Braz. j. med. biol. res ; 38(11): 1643-1647, Nov. 2005.
Article in English | LILACS | ID: lil-414716

ABSTRACT

To evaluate the human T-cell lymphotropic virus type I (HTLV-I) proviral DNA load among asymptomatic HTLV-I-infected carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), real time PCR using TaqMan probes for the pol gene was performed in two million peripheral blood mononuclear cells (PBMC). The albumin gene was the internal genomic control and MT2 cells were used as positive control. The results are reported as copies/10,000 PBMC, and the detection limit was 10 copies. A total of 89 subjects (44 HAM/TSP and 45 healthy HTLV-I-infected carriers) followed up at the Institute of Infectious Diseases "Emilio Ribas" and in the Neurology Division of Hospital of Clínicas were studied. The asymptomatic HTLV-I-infected carriers had a median number of 271 copies (ranging from 5 to 4756 copies), whereas the HAM/TSP cases presented a median of 679 copies (5-5360 copies) in 10,000 PBMC. Thus, HAM/TSP patients presented a significantly higher HTLV-I proviral DNA load than healthy HTLV-I carriers (P = 0.005, one-way Mann-Whitney test). As observed in other persistent infections, proviral DNA load quantification may be an important tool for monotoring HTLV-I-infected subjects. However, long-term follow-up is necessary to validate this assay in the clinical setting.


Subject(s)
Humans , DNA, Viral/analysis , Paraparesis, Tropical Spastic/virology , Proviruses/genetics , Human T-lymphotropic virus 1/genetics , Case-Control Studies , DNA, Viral/genetics , DNA, Viral/immunology , Leukocytes, Mononuclear/immunology , Polymerase Chain Reaction , Paraparesis, Tropical Spastic/immunology , Proviruses/immunology , Viral Load , Human T-lymphotropic virus 1/immunology
12.
FEMS Microbiol Lett ; 253(2): 267-72, 2005 Dec 15.
Article in English | MEDLINE | ID: mdl-16243455

ABSTRACT

The production of hemicellulose and cellulose degrading enzymes by the white-rot fungus Ceriporiopsis subvermispora was determined while growing in Pinus taeda wood chips. Enzymes produced by the fungus were extracted after 30 days of cultivation and at least two different xylanases were secreted. An endo-(1,4)-beta-xylanase was purified by means of ultrafiltration, anion exchange chromatography and gel filtration. Its molecular mass was 29 kDa and the pH and temperature optima were 5.0 and 60 degrees C, respectively. The endo-xylanase was able to hydrolyze xylan to principally xylotriose and xylotetraose and it has different activities against different xylans. With birchwood xylan as substrate, the enzyme showed a K(m) of 1.93 mg/ml and specific activity of 538 units/mg protein at 50 degrees C.


Subject(s)
Polyporales/enzymology , Xylosidases/classification , Xylosidases/metabolism , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Hydrolysis , Pinus taeda/microbiology , Xylosidases/isolation & purification
13.
Lett Appl Microbiol ; 40(4): 283-8, 2005.
Article in English | MEDLINE | ID: mdl-15752219

ABSTRACT

AIMS: To develop strategies for increasing the growth of Lentinula edodes in eucalyptus residues. To this end, we have examined the effects of cereal brans additions on production of mycelial biomass and enzymes. METHODS AND RESULTS: Three isolates of the mushroom shiitake, L. edodes (Berk. Pegler), were evaluated for enzyme and ergosterol production on eucalyptus residue supplemented with 5, 10, 15 and 20% (w/w) of soya, wheat or rice brans. Nitrogen imput on eucalyptus residues accelerated mycelial growth by supplying the L. edodes with this limiting nutrient. High levels of enzymes activities were produced in eucalyptus residues supplemented by soya bran. Comparison of cellulose and xylanase production with manganese peroxidase (MnP) at 20% soya bran indicated that hydrolytic enzymes, but oxidative enzymes were reduced. CONCLUSIONS: Mycelial growth measurements revealed that eucalyptus residues supplemented with cereal brans supported fast growth of L. edodes, indicating that mycelium extension is related to the bioavailability of nitrogen. The type and concentration of nutrient supplement has a considerable effect both on substrate colonization and on the type of hydrolytic and oxidative enzymes produced. These characteristics may be useful for mushroom growing. SIGNIFICANCE AND IMPACT OF THE STUDY: Lentinula edodes is commercially important for edible mushroom production and supplements which enhance growth and enzymes production might also be beneficial for mushroom yields.


Subject(s)
Edible Grain/metabolism , Eucalyptus/microbiology , Shiitake Mushrooms/growth & development , Shiitake Mushrooms/metabolism , Biomass , Cellulase/analysis , Dietary Fiber/metabolism , Endo-1,4-beta Xylanases/analysis , Ergosterol/analysis , Ergosterol/biosynthesis , Mycelium/growth & development , Nitrogen/metabolism , Oryza/metabolism , Peroxidases/analysis , Glycine max/metabolism
14.
Br J Dermatol ; 149(4): 776-81, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14616369

ABSTRACT

BACKGROUND: Patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy frequently display cutaneous alterations such as acquired ichthyosis. OBJECTIVES: Elucidation of the pattern of acquired ichthyosis in HTLV-I-associated myelopathy. METHODS: Skin fragments from 10 patients with HTLV-I-associated myelopathy presenting with acquired ichthyosis were assessed by histopathological and immunohistochemical tests. We used anticytokeratin antibodies related to normal keratinization (K1/K10), and others related to cutaneous conditions such as activation, migration and hyperproliferation of keratinocytes (K6/K16), and involucrin, a precursor protein in the formation of the protein envelope in keratinocytes. For quantification of the proliferating basal and parabasal cells the anti-Ki-67 antibody was employed. RESULTS: On light microscopy, all skin specimens displayed orthokeratotic hyperkeratosis and hypogranulosis. Three of them presented focal parakeratosis. A slight to moderate perivascular infiltrate of mononuclear lymphocytes was observed in seven cases, three of which showed discrete spongiosis with epidermotropism of lymphocytes. All fragments displayed coexpression of K1, K10 and K16 in the suprabasal layers. Expression of involucrin was also observed in all cases, in the upper spinous and granular layers. Focal expression of K6 was observed in three cases, under a parakeratotic area. The mean number of Ki-67+ basal and parabasal cells was 3.5 cells per mm, similar to that in control skin. CONCLUSIONS: In acquired ichthyosis related to HTLV-I-associated myelopathy, histopathology revealed orthokeratotic hyperkeratosis and a perivascular inflammatory infiltrate of mononuclear lymphocytes, with areas of parakeratosis and foci of epidermotropism in rare cases. The expression profiles of K1, K10 and involucrin were similar to those in normal skin. The diffuse coexpression of K16 with K1 and K10 throughout the analysed epidermis, as well as the occurrence of restricted areas of parakeratosis expressing K6, indicate the presence of keratinocyte activation with induction of the alternative keratinization pathway, probably dependent on the cytokines liberated by the mononuclear cells of the dermal inflammatory infiltrate infected with HTLV-I. The absence of acanthosis and of increased cellular kinetics, as shown by the low rate of Ki-67 antigen expression, allow the inference that the pattern of acquired ichthyosis related to HTLV-I-associated myelopathy may be retentional. The observation of foci of parakeratosis expressing K6 in three specimens suggests that, at least in certain areas and in some cases, interference with epidermal differentiation and maturation occurs.


Subject(s)
Ichthyosis/virology , Paraparesis, Tropical Spastic/complications , Cell Division , Humans , Ichthyosis/pathology , Keratosis/pathology , Keratosis/virology , Ki-67 Antigen/metabolism , Leg Dermatoses/pathology , Leg Dermatoses/virology
15.
Lett Appl Microbiol ; 36(3): 177-81, 2003.
Article in English | MEDLINE | ID: mdl-12581379

ABSTRACT

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.


Subject(s)
Agar/chemistry , Aspergillus/metabolism , Culture Media/chemistry , Hydroxybenzoates/chemistry , Siderophores/biosynthesis , Aspergillus/classification , Aspergillus/growth & development , Hydrogen-Ion Concentration , Iron/metabolism , Siderophores/metabolism
16.
Enzyme Microb Technol ; 28(6): 522-526, 2001 Apr 05.
Article in English | MEDLINE | ID: mdl-11267647

ABSTRACT

Xylanase, oxidative enzymes and iron-binding compounds were detected in the filtrates of Wolfiporia cocos and Poria medula-panis grown in wheat bran liquid medium. Xylanase and iron-binding compounds were produced at high levels by the brown-rot fungus (BR) W. cocos and at low levels by the white-rot fungus (WR) P. medula-panis. Phenoloxidase was produced only by P. medula-panis. Polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) showed a wide variety of bands for extracellular proteins produced by W.cocos, with low molecular weight (<30 kDa) and minor bands with molecular weight above 45 kDa. Two bands with xylanase activity derived from W. cocos extracts were detected in the gels, whereas many different bands with xylanase activity were found in the extracts from P. medula-panis. P. medula-panis is a selective lignin degrader, whereas W. cocos preferentially removes cellulose from wood.

17.
J Ind Microbiol Biotechnol ; 23(1): 682-5, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10455501

ABSTRACT

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45 degrees C and was stable at 50 degrees C and pH 5.0 up to 4 h xylanase. XYL2 had a low Km with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp.


Subject(s)
Trichoderma/enzymology , Xylosidases/isolation & purification , Amino Acids/analysis , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Ultrafiltration , Wood , Xylan Endo-1,3-beta-Xylosidase , Xylosidases/chemistry , Xylosidases/metabolism
18.
Braz J Med Biol Res ; 32(8): 947-52, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10454755

ABSTRACT

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28 degree C in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ss-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20. 45 cp. The efficiency of delignification was 33%.


Subject(s)
Cell Culture Techniques/methods , Trichoderma/enzymology , Xylans/metabolism , Xylosidases/metabolism , Xylosidases/isolation & purification
19.
Braz. j. med. biol. res ; 32(8): 947-52, Aug. 1999.
Article in English | LILACS | ID: lil-238962

ABSTRACT

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28oC in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ß-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20.45 cp. The efficiency of delignification was 33


Subject(s)
Cell Culture Techniques/methods , Trichoderma/enzymology , Xylans/metabolism , Xylosidases/metabolism , Xylosidases/isolation & purification
20.
J Microbiol Methods ; 37(1): 1-6, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10395458

ABSTRACT

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.


Subject(s)
Bacteria/metabolism , Culture Media/chemistry , Fungi/metabolism , Hydroxybenzoates/chemistry , Indicators and Reagents/chemistry , Siderophores/metabolism , Agar , Bacteria/growth & development , Fungi/growth & development , Microbiological Techniques
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