Effective In Vitro Control of Two Phytopathogens of Agricultural Interest Using Cell-Free Extracts of Pseudomonas fluorescens and Chitosan.

A biofungicide is a natural product that can be derived from various sources such as, among others, microorganisms, higher plants, animal products, phytochemicals, semiochemicals, and antagonist microorganisms. One of the most important approaches for the production of biofungicides is the combination of biocontrol agents. This study showed the inhibition growth of Alternaria alternata and Fusarium solani treated with cell-free extracts of P. fluorescens. Using thin-layer chromatography and plate assays it was also demonstrated that the cell-free extracts of P. fluorescens contained siderophores and derivates of 4-diacetylphloroglucinol and phenazine. Moreover, the combination of cell-free extracts of P. fluorescens and chitosan [50-1.5% (v/v)] had a synergistic effect since they notably inhibited the mycelial growth of A. altenata and F. solani. Various morphological alterations to the mycelia and conidia of the treated fungi as a result of this combination were also observed. The present study could be a starting point to control other fungal phytopathogens using different cell-free extracts and chitosan as biocontrol agents.

In vitro biosynthesis of Ag, Au and Te-containing nanostructures by Exiguobacterium cell-free extracts.

BACKGROUND: The bacterial genus Exiguobacterium includes several species that inhabit environments with a wide range of temperature, salinity, and pH. This is why the microorganisms from this genus are known generically as polyextremophiles. Several environmental isolates have been explored and characterized for enzyme production as well as for bioremediation purposes. In this line, toxic metal(loid) reduction by these microorganisms represents an approach to decontaminate soluble metal ions via their transformation into less toxic, insoluble derivatives. Microbial-mediated metal(loid) reduction frequently results in the synthesis of nanoscale structures-nanostructures (NS) -. Thus, microorganisms could be used as an ecofriendly way to get NS. RESULTS: We analyzed the tolerance of Exiguobacterium acetylicum MF03, E. aurantiacum MF06, and E. profundum MF08 to Silver (I), gold (III), and tellurium (IV) compounds. Specifically, we explored the ability of cell-free extracts from these bacteria to reduce these toxicants and synthesize NS in vitro, both in the presence or absence of oxygen. All isolates exhibited higher tolerance to these toxicants in anaerobiosis. While in the absence of oxygen they showed high tellurite- and silver-reducing activity at pH 9.0, whereas AuCl4- which was reduced at pH 7.0 in both conditions. Given these results, cell-free extracts were used to synthesize NS containing silver, gold or tellurium, characterizing their size, morphology and chemical composition. Silver and tellurium NS exhibited smaller size under anaerobiosis and their morphology was circular (silver NS), starred (tellurium NS) or amorphous (gold NS). CONCLUSIONS: This nanostructure-synthesizing ability makes these isolates interesting candidates to get NS with biotechnological potential.

Biomimetic Approaches for the Development of New Antifouling Solutions: Study of Incorporation of Macroalgae and Sponge Extracts for the Development of New Environmentally-Friendly Coatings.

Biofouling causes major economic losses in the maritime industry. In our site study, the Bay of La Paz (Gulf of California), biofouling on immersed structures is a major problem and is treated mostly with copper-based antifouling paints. Due to the known environmental effect of such treatments, the search for environmentally friendly alternatives in this zone of high biodiversity is a priority to ensure the conservation and protection of species. The aim of this work was to link chemical ecology to marine biotechnology: indeed, the natural defense of macroalgae and sponge was evaluated against biofoulers (biofilm and macrofoulers) from the same geographical zone, and some coatings formulation was done for field assays. Our approach combines in vitro and field bioassays to ensure the selection of the best AF agent prospects. The 1st step consisted of the selection of macroalgae (5 species) and sponges (2 species) with surfaces harboring a low level of colonizers; then extracts were prepared and assayed for toxicity against Artemia, activity towards key marine bacteria involved in biofilm formation in the Bay of La Paz, and the potency to inhibit adhesion of macroorganisms (phenoloxidase assays). The most active and non-toxic extracts were further studied for biofouling activity in the adhesion of the bacteria involved in biofilm formation and through incorporation in marine coatings which were immersed in La Paz Bay during 40 days. In vitro assays demonstrated that extracts of Laurencia gardneri, Sargassum horridum (macroalgae), Haliclona caerulea and Ircinia sp. (sponges) were the most promising. The field test results were of high interest as the best formulation were composed of extracts of H. caerulea and S. horridum and led to a reduction of 32% of biofouling compared with the control.

Proteomic Analysis Reveals Allergen Variability among Breeds of the Dust Mite Blomia tropicalis.

BACKGROUND: The dawn of the "omics" technologies has changed allergy research, increasing the knowledge and identification of new allergens. However, these studies have been almost restricted to Dermatophagoides spp. Although Blomia tropicalis has long been established as a clinically important source of allergens, a thorough proteomic characterization is still lacking for this dust mite. OBJECTIVE: To increase knowledge of B. tropicalis allergens through proteomic analysis. METHODS: Eleven in-bred lineages of B. tropicalis were obtained from 11 unique different pregnant females. Their somatic extracts were analyzed and compared with a commercially available extract by liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Considerable differences in the protein expression profiles were found among the breeds, and most of them displayed higher expression levels of major allergens than the commercially available extract. Blo t 2 was the most prominent allergenic protein in the analyzed extracts. Six identified allergens and 14 isoforms have not yet been recognized by IUIS. Conversely, 3 previously recognized B. tropicalis allergens were not found. CONCLUSIONS: The clear impact of inbreeding on allergen content shown by our study leads us to conclude that the quantification and/or identification of allergens from in-bred lines should be routinely considered for mite cultivation in order to select breeds with higher amounts of major allergens. In this sense, LC-MS/MS may be a useful method to achieve this quality control for research and commercial purposes.

Influence of Eugenia uniflora Extract on Adhesion to Human Buccal Epithelial Cells, Biofilm Formation, and Cell Surface Hydrophobicity of Candida spp. from the Oral Cavity of Kidney Transplant Recipients.

This study evaluated the influence of the extract of Eugenia uniflora in adhesion to human buccal epithelial cells (HBEC) biofilm formation and cell surface hydrophobicity (CSH) of Candida spp. isolated from the oral cavity of kidney transplant patients. To evaluate virulence attributes in vitro, nine yeasts were grown in the presence and absence of 1000 µg/mL of the extract. Adhesion was quantified using the number of Candida cells adhered to 150 HBEC determined by optical microscope. Biofilm formation was evaluated using two methodologies: XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and crystal violet assay, and further analyzed by electronic scan microscopy. CSH was quantified with the microbial adhesion to hydrocarbons test. We could detect that the extract of E. uniflora was able to reduce adhesion to HBEC and CSH for both Candida albicans and non-Candida albicansCandida species. We also observed a statistically significant reduced ability to form biofilms in biofilm-producing strains using both methods of quantification. However, two highly biofilm-producing strains of Candida tropicalis had a very large reduction in biofilm formation. This study reinforces the idea that besides growth inhibition, E. uniflora may interfere with the expression of some virulence factors of Candida spp. and may be possibly applied in the future as a novel antifungal agent.

Application of aqueous two-phase micellar system to improve extraction of adenoviral particles from cell lysate.

Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two-phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK-293) cells to a concentration of 1.4 × 1010 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X-114 in a 23 full factorial design with center points. The systems were formed with Triton X-114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37 °C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X-114, a cell lysate pH of 7.0, and an incubation temperature at 33 °C, yielding 3.51 × 1010 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3-fold, purifying it by 2.2-fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two-phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps.

Physicochemical Characteristics of Transferon™ Batches.

Transferon, a biotherapeutic agent that has been used for the past 2 decades for diseases with an inflammatory component, has been approved by regulatory authorities in Mexico (COFEPRIS) for the treatment of patients with herpes infection. The active pharmaceutical ingredient (API) of Transferon is based on polydispersion of peptides that have been extracted from lysed human leukocytes by a dialysis process and a subsequent ultrafiltration step to select molecules below 10 kDa. To physicochemically characterize the drug product, we developed chromatographic methods and an SDS-PAGE approach to analyze the composition and the overall variability of Transferon. Reversed-phase chromatographic profiles of peptide populations demonstrated batch-to-batch consistency from 10 representative batches that harbored 4 primary peaks with a relative standard deviation (RSD) of less than 7%. Aminogram profiles exhibited 17 proteinogenic amino acids and showed that glycine was the most abundant amino acid, with a relative content of approximately 18%. Further, based on their electrophoretic migration, the peptide populations exhibited a molecular mass of about 10 kDa. Finally, we determined the Transferon fingerprint using a mass spectrometry tool. Because each batch was produced from independent pooled buffy coat samples from healthy donors, supplied by a local blood bank, our results support the consistency of the production of Transferon and reveal its peptide identity with regard to its physicochemical attributes.

CdSe magic-sized quantum dots incorporated in biomembrane models at the air-water interface composed of components of tumorigenic and non-tumorigenic cells.

Cadmium selenide (CdSe) magic-sized quantum dots (MSQDs) are semiconductor nanocrystals with stable luminescence that are feasible for biomedical applications, especially for in vivo and in vitro imaging of tumor cells. In this work, we investigated the specific interaction of CdSe MSQDs with tumorigenic and non-tumorigenic cells using Langmuir monolayers and Langmuir-Blodgett (LB) films of lipids as membrane models for diagnosis of cancerous cells. Surface pressure-area isotherms and polarization modulation reflection-absorption spectroscopy (PM-IRRAS) showed an intrinsic interaction between the quantum dots, inserted in the aqueous subphase, and Langmuir monolayers constituted either of selected lipids or of tumorigenic and non-tumorigenic cell extracts. The films were transferred to solid supports to obtain microscopic images, providing information on their morphology. Similarity between films with different compositions representing cell membranes, with or without the quantum dots, was evaluated by atomic force microscopy (AFM) and confocal microscopy. This study demonstrates that the affinity of quantum dots for models representing cancer cells permits the use of these systems as devices for cancer diagnosis.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a.

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae Euglena tuba.

BACKGROUND: Unstable generation of free radicals in the body are responsible for many degenerative diseases. A bloom forming algae Euglena tuba growing abundantly in the aquatic habitats of Cachar district in the state of Assam in North-East India was analysed for its phytochemical contents, antioxidant activity as well as free radical scavenging potentials. RESULTS: Based on the ability of the extract in ABTS•+ radical cation inhibition and Fe3+ reducing power, the obtained results revealed the prominent antioxidant activity of the algae, with high correlation coefficient of its TEAC values to the respective phenolic and flavonoid contents. The extract had shown its scavenging activity for different free radicals and 41.89 ± 0.41 µg/ml, 5.83 ± 0.07 µg/ml, 278.46 ± 15.02 µg/ml and 223.25 ± 4.19 µg/ml were determined as the IC50 values for hydroxyl, superoxide, nitric oxide and hypochlorous acid respectively, which are lower than that of the corresponding reference standards. The phytochemical analysis also revealed that the phenolics, flavonoids, alkaloids, tannins and carbohydrates are present in adequate amount in the extract which was confirmed by HPLC analysis. CONCLUSIONS: The results showed that 70% methanol extract of the algae possesses excellent antioxidant and free radical scavenging properties.

Assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae Euglena tuba

BACKGROUND: Unstable generation of free radicals in the body are responsible for many degenerative diseases. A bloom forming algae Euglena tuba growing abundantly in the aquatic habitats of Cachar district in the state of Assam in North-East India was analysed for its phytochemical contents, antioxidant activity as well as free radical scavenging potentials. RESULTS: Based on the ability of the extract in ABTS•+ radical cation inhibition and Fe3+ reducing power, the obtained results revealed the prominent antioxidant activity of the algae, with high correlation coefficient of its TEAC values to the respective phenolic and flavonoid contents. The extract had shown its scavenging activity for different free radicals and 41.89 ± 0.41 µg/ml, 5.83 ± 0.07 µg/ml, 278.46 ± 15.02 µg/ml and 223.25 ± 4.19 µg/ml were determined as the IC50 values for hydroxyl, superoxide, nitric oxide and hypochlorous acid respectively, which are lower than that of the corresponding reference standards. The phytochemical analysis also revealed that the phenolics, flavonoids, alkaloids, tannins and carbohydrates are present in adequate amount in the extract which was confirmed by HPLC analysis. CONCLUSIONS: The results showed that 70% methanol extract of the algae possesses excellent antioxidant and free radical scavenging properties.

Identification of Alternaria alternata mycotoxins by LC-SPE-NMR and their cytotoxic effects to soybean (Glycine max) cell suspension culture.

This present work describes the application of liquid chromatography-solid phase extraction-nuclear magnetic resonance spectroscopy to analyse Alternaria alternata crude extracts. Altenusin (1), alternariol (2), 3'-hydroxyalternariol monomethyl ether (3), and alternariol monomethyl ether (4), were separated and identified. High-resolution mass spectrometry confirmed the proposed structures. The cytotoxic effects of these compounds towards plants were determined using soybean (Glycine max) cell cultures as a model. EC(50) values which range from 0.11 (± 0.02) to 4.69 (± 0.47) µM showed the high cytotoxicity of these compounds.

Antibacterial activity and cytotoxicity analysis of halistanol trisulphate from marine sponge Petromica citrina.

OBJECTIVES: An aqueous extract and fraction from the marine sponge Petromica citrina have antibacterial activity. We performed a chemical and biological characterization of the antibiotic substance from P. citrina and investigated its mode of action on Staphylococcus aureus cells. METHODS: The inhibitory activity of the aqueous extract of P. citrina was determined against 14 bacteria belonging to type strains and clinical antibiotic-resistant strains. The aqueous extract was fractionated under bioassay guidance and the bioactive substance was identified by its (1)H-NMR, (13)C-NMR and mass spectra. The MIC and the MBC of this substance were determined. This substance was also subjected to cytotoxic bioassays. The mode of action on S. aureus cells was investigated by light and transmission electron microscopy analysis. RESULTS: P. citrina showed a large spectrum of activity against type strains and resistant-bacteria such as S. aureus, Staphylococcus epidermidis, Enterococcus faecalis, Mycobacterium fortuitum and Neisseria gonorrhoeae. The aqueous extract was fractionated and halistanol trisulphate (24ε,25-dimethylcholestane-2ß,3α,6α-triol trisodium sulphate) was isolated for the first time from P. citrina. Halistanol trisulphate had a bactericidal effect on exponentially growing S. aureus cells at the MIC (512 mg/L). Cytotoxicity biossays showed moderate toxicity against cancer cell line L929 (fibrosarcoma). This substance apparently acts by damaging the cell membrane, with subsequent cell lysis. CONCLUSIONS: Halistanol trisulphate is a broad-spectrum antibiotic isolated from P. citrina with a mode of action involving disruption of the cytoplasmic membrane. It is a new candidate for research on antibacterial substances.

Neutralization of Pseudomonas aeruginosa enzymatic activity by antibodies elicited with proteins of Larrea divaricata Cav.

Larrea divaricata Cav. (Jarilla) is a bush widely used in folk therapy for the treatment of several pathologies. Partially purified proteins of crude extract (JPCE) cross-react with proteins of Gram-negative bacteria, including Pseudomonas aeruginosa, which is an opportunistic pathogen that causes several intrahospitalary infections. This bacterium produces many proteins with enzymatic activity, including hemolysins and proteases that play a major role in acute infection caused by this bacterium. The aim of our work was to investigate if antibodies against with L. divaricata neutralize the hemolytic and proteolytic activity of P. aeruginosa. The hemolytic activity of soluble cellular proteins was inhibited 100% and extracellular proteins (EP) showed an inhibition between 44 and 95% when both bacterial fractions were treated with anti-JPCE serum. Also, in EP the neutralization was directed towards the active site of the hemolysin. When protease activity of extracellular products was tested, bands of 217, 155, 121, 47 and 27 kDa were observed in native zymograms. Neutralization between 55 and 70% of the bands of 217, 155 and 121 kDa was observed when EP were treated with anti-JPCE serum. In conclusion, our data clearly demonstrate that antibodies elicited with L. divaricata' proteins are able to neutralize the hemolytic and proteolytic activity of P. aeruginosa cellular and extracellular proteins. Our study constitutes the first report that associates the immunogenicity of plant proteins and bacterial proteins with enzymatic activity. These findings could be relevant in the development of alternatives therapies for patients suffering intrahospitalary opportunistic infections with P. aeruginosa.

Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein.

BACKGROUND AND AIMS: Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. METHODS: Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. RESULTS: After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. CONCLUSIONS: The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway.

Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

Characterization and antineoplasic effect of extracts obtained from Pleurotus sajor-caju fruiting bodies.

Fungi of the Pleurotus genus present a great industrial interest due to their possibility of producing pharmacological compounds, pigments, aromas, organic acids, polysaccharides, enzymes, vitamins, amino acids, etc. Among the therapeutic products, we can highlight those with antineoplasic activity, attributed to the fungi cell wall components. Based on this, the objective of this work was to study the antineoplasic capacity of the polysaccharidic fractions obtained from Pleurotus sajor-caju fruiting bodies. Female Swiss mice were inoculated with the Ehrlich ascitic tumor (5 x 10(6) cells/animal) in ascitic form. The polysaccharidic fractions were administered intraperitoneally, during a 6-day period. Fractions FI and FII presented a lower volume of ascitic liquid (3.1 and 1.8 mL, respectively) and a higher reduction in the number of neoplasic cells present in the ascitic liquid (86.2% and 85%, respectively), when compared to the positive control (group inoculated with the tumor but without treatment). These fractions were characterized in terms of monosaccharide composition. Glucose was the major component detected, followed by galactose and mannose. The anomeric carbon configuration of the beta-glucan was confirmed by the (13)C NMR (delta 103.7). Substituted and free C3 and C6 were also detected. Protein bands were confirmed through infrared analysis.

Nitrogen source optimization for cellulase production by Penicillium funiculosum, using a sequential experimental design methodology and the desirability function.

The present study aimed at maximizing cellulase production by Penicillium funiculosum using sequential experimental design methodology for optimizing the concentrations of nitrogen sources. Three sequential experimental designs were performed. The first and the second series of experiments consisted of a 2(4) and a 2(3) factorial designs, respectively, and in the third one, a central composite rotational design was used for better visualizing the optimum conditions. The following nitrogen sources were evaluated: urea, ammonium sulfate, peptone, and yeast extract. Peptone and ammonium sulfate were removed from the medium optimization since they did not present significant statistical effect on cellulase production. The optimal concentrations of urea and yeast extract predicted by the model were 0.97 and 0.36 g/L, respectively, which were validated experimentally. By the use of the desirability function, it was possible to maximize the three main enzyme activities simultaneously, which resulted in values for FPase of 227 U/L, for CMCase of 6,917 U/L, and for beta-glucosidase of 1,375 U/L. These values corresponded to increases of 3.3-, 3.2-, and 6.7-folds, respectively, when compared to those obtained in the first experimental design. The results showed that the use of sequential experimental designs associated to the use of the desirability function can be used satisfactorily to maximize cellulase production by P. funiculosum.

Dynamics of heparin-binding proteins on boar sperm.

The presence, topology and dynamics of heparin-binding proteins (HBP) on boar sperm were evaluated. HBP distribution was analyzed by subcellular parting, using biotinylated heparin followed by colorimetric detection. HBP were detected as peripherical and integral periacrosomal membrane proteins. Indirect fluorescence microscopy of sperm incubated with biotinylated heparin was used to evidence heparin binding on sperm at different physiological stages. Two different fluorescent patterns (A and B) were found, which probably correspond to non-capacitated and capacitated sperm as assessed by the ability to undergo acrosome reaction with calcium ionophore A23187 and by the increase of p32 phosphorylated protein. In A pattern, corresponding to untreated sperm, fluorescence located mostly on the post-acrosomal region; in B pattern, corresponding to incubated sperm, on the acrosomal region. Upon incubation under capacitating conditions (TALP), sperm having the B pattern was augmented compared with non-incubated sperm (p<0.001). Differences in the HBP patterns (p<0.0001) were observed in sperm incubated under non-capacitating conditions in relation to sperm incubated in TALP, indicating that the modification of HBP patterns is probably related to capacitation. No difference was observed when untreated sperm were permeabilized prior to staining, suggesting that HBP are present on the sperm surface. The effect of heparin on capacitation dependent protein tyrosine phosphorylation was also analyzed, finding a decrease in p32 phosphorylation in the presence of heparin. This suggests that the capacitation enhancement mediated by this glycosaminoglycan involves an alternative intracellular pathway. The finding that heparin binds to sperm differently according to its physiological state, is a new evidence of the remodelling of sperm membrane surface upon capacitation and may provide a useful and relatively simple method to evaluate in vitro modification of boar sperm physiological state.