The combination of ACE I/D and ACE2 G8790A polymorphisms revels susceptibility to hypertension: A genetic association study in Brazilian patients.

BACKGROUND: Systemic arterial hypertension (SAH) is a multifactorial condition that already affects one third of the worldwide population. The identification of candidate genes for hypertension is a challenge for the next years. Nevertheless, the small contribution of each individual genetic factor to the disease brings the necessity of evaluate genes in an integrative manner and taking into consideration the physiological interaction of functions. Angiotensin I-converting enzymes, ACE and ACE2, are key regulators of blood pressure that have counterbalance roles by acting on vasoactive peptides from Renin-Angiotensin-Aldosterone System (RAAS). Insertion/deletion (I/D) polymorphism of ACE gene and single nucleotide polymorphism G8790A of ACE2 gene have been associated with susceptibility to SAH, but the literature is controversial. We proposed to evaluate these two polymorphisms jointly exploring the combined effects of ACE and ACE2 genotypes on SAH susceptibility, an approach that have not been done yet for ACE and ACE2 polymorphisms. METHODS AND FINDINGS: This genetic association study included 117 hypertensive (mean age 59.7 years) patients and 123 normotensive and diabetes-free controls (mean age 57.5 years). ACE and ACE2 polymorphisms were genotyped by SYBR Green real-time PCR and RFLP-PCR, respectively. Crude and adjusted odds ratio (OR) values were calculated to estimate the susceptibility to SAH development. It was obtained homogeneity regarding distribution by sex, age range, smoking, alcohol consumption and body mass index (BMI) between case and control groups. No-association was verified for each gene individually, but the combination of ACE and ACE2 polymorphisms on female gender revealed a significative association for DD/G_ carriers who had a 3-fold increased risk to SAH development (p = 0.03), with a stronger susceptibility on DD/GG carriers (7-fold increased risk, p = 0.01). The D allele of ACE showed association with altered levels of lipid profile variables on case group (VLDL-cholesterol, p = 0.01) and DD genotype in all individuals analysis (triglycerides, p = 0.01 and VLDL-cholesterol, p = 0.01). CONCLUSION: These findings indicate that the combination of ACE and ACE2 polymorphisms effects may play a role in SAH predisposition been the DD/G_ genotype the susceptibility profile. This result allowed us to raise the hypothesis that an increased activity of ACE (prohypertensive effects) in conjunction with reduced ACE2 activity (antihypertensive effects) could be the underlining mechanism. The association of ACE D allele with lipid alterations indicate that this can be a marker of poor prognostic on SAH evolution and contribute to CVD development. Although these preliminary findings must be confirmed by further researches with larger sample size, we could observe that the integrative analysis of ACE and ACE2 can be an informative tool in hypertension understanding that needs to be explored in new studies.

Trichoderma/pathogen/plant interaction in pre-harvest food security.

Large losses before crop harvesting are caused by plant pathogens, such as viruses, bacteria, oomycetes, fungi, and nematodes. Among these, fungi are the major cause of losses in agriculture worldwide. Plant pathogens are still controlled through application of agrochemicals, causing human disease and impacting environmental and food security. Biological control provides a safe alternative for the control of fungal plant pathogens, because of the ability of biocontrol agents to establish in the ecosystem. Some Trichoderma spp. are considered potential agents in the control of fungal plant diseases. They can interact directly with roots, increasing plant growth, resistance to diseases, and tolerance to abiotic stress. Furthermore, Trichoderma can directly kill fungal plant pathogens by antibiosis, as well as via mycoparasitism strategies. In this review, we will discuss the interactions between Trichoderma/fungal pathogens/plants during the pre-harvest of crops. In addition, we will highlight how these interactions can influence crop production and food security. Finally, we will describe the future of crop production using antimicrobial peptides, plants carrying pathogen-derived resistance, and plantibodies.

Effect of vitamin E in ovo feeding to broiler embryos on hatchability, chick quality, oxidative state, and performance.

The objective of the present study was to ascertain the effect of in ovo feeding of vitamin E (VE) on the incubation results, quality, and oxidative state of newborn chicks and on the initial performance results. The design consisted of randomized blocks with treatments at different levels of VE (0.0, 27.5, 38.5, 49.5, and 60.4 IU). On 17.5 d of embryonic development, 780 eggs underwent in ovo injection using a manual needle. VE supplementation of 60.4 IU provided the highest hatching rate (P < 0.05) and shortest hatch window (P < 0.05). Better results regarding chick physical quality were observed in groups supplemented with VE (body weight, length, newborn chick quality score) and higher chick weight/egg weight ratios (P < 0.05). VE inoculation did not have any effect on the chicks' immunological system (P > 0.05). Greater development of the small intestine (intestine weight/yolk free chick weight and higher villi in duodenum) and better feed conversion over all periods studied (1 to 7, 1 to 14, and 1 to 21 d) were observed among chicks that received in ovo VE supplementation (P < 0.05). The total protein concentrations in the liver and striated breast skeletal muscle tissue were highest in chicks that received 60.4 IU of VE (P < 0.05). The highest catalase activity was observed in the livers of newborn chicks supplemented with 60.4 IU of VE (P < 0.05). It was concluded that in ovo VE supplementation improved the chicks' oxidative state, which led to improvements in incubation results, chick quality and performance results.

Involvement of Trichoderma harzianum Epl-1 Protein in the Regulation of Botrytis Virulence- and Tomato Defense-Related Genes.

Several Trichoderma spp. are well known for their ability to: (i) act as important biocontrol agents against phytopathogenic fungi; (ii) function as biofertilizers; (iii) increase the tolerance of plants to biotic and abiotic stresses; and (iv) induce plant defense responses via the production and secretion of elicitor molecules. In this study, we analyzed the gene-regulation effects of Trichoderma harzianum Epl-1 protein during the interactions of mutant Δepl-1 or wild-type T. harzianum strains with: (a) the phytopathogen Botrytis cinerea and (b) with tomato plants, on short (24 h hydroponic cultures) and long periods (4-weeks old plants) after Trichoderma inoculation. Our results indicate that T. harzianum Epl-1 protein affects the in vitro expression of B. cinerea virulence genes, especially those involved in the botrydial biosynthesis (BcBOT genes), during the mycoparasitism interaction. The tomato defense-related genes were also affected, indicating that Epl-1 is involved in the elicitation of the salicylic acid pathway. Moreover, Epl-1 also regulates the priming effect in host tomato plants and contributes to enhance the interaction with the host tomato plant during the early stage of root colonization.

Growth and expression of relevant metabolic genes of Clostridium thermocellum cultured on lignocellulosic residues.

The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.

Peptaibols from Trichoderma asperellum TR356 strain isolated from Brazilian soil.

The Trichoderma genus consists of a group of free-living filamentous fungi, including species able to act as biological control agents (BCAs) against pathogenic fungi. It is believed that this ability is due to synergy between several mechanisms, including the production of a wide variety of secondary metabolites by these organisms. Among these, we highlight the production of peptaibols, an antibiotic peptide group characterized by the presence of non-proteinogenic amino acids such as α-aminoisobutyrate (Aib), as well as by N-terminal modifications and amino alcohols in the C-terminal region. This study aimed to outline a profile of peptaibol production and to identify secreted peptaibols from the Trichoderma asperellum TR356 strain, described as an efficient BCA against S. sclerotiorum. The fungus was grown on TLE 0.3% glucose medium for 5 days, with agitation at 120 rpm in the dark. Liquid medium filtrate was used as the metabolite source. These extracts were subjected to high performance liquid chromatography (HPLC) and subsequent analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF). The results indicate the production of two classes of peptaibols for this T. asperellum strain. Primary structures of two asperelines (A and E) and five trichotoxins (T5D2, T5E, T5F, T5G and 1717A) have been elucidated. Most of these peptaibols had been previously described in T. viride and T. asperellum marine strains. This is the first report of some of these compounds being produced by a T. asperellum strain from soil. Future analyses will be necessary to elucidate the three-dimensional structures and their activities against pathogens.

Analysis of Phaseolus vulgaris response to its association with Trichoderma harzianum (ALL-42) in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani.

The present study was carried out to evaluate the ability of Trichoderma harzianum (ALL 42-isolated from Brazilian Cerrado soil) to promote common bean growth and to modulate its metabolism and defense response in the presence or absence of the phytopathogenic fungi Rhizoctonia solani and Fusarium solani using a proteomic approach. T. harzianum was able to promote common bean plants growth as shown by the increase in root/foliar areas and by size in comparison to plants grown in its absence. The interaction was shown to modulate the expression of defense-related genes (Glu1, pod3 and lox1) in roots of P. vulgaris. Proteomic maps constructed using roots and leaves of plants challenged or unchallenged by T. harzianum and phytopathogenic fungi showed differences. Reference gels presented differences in spot distribution (absence/presence) and relative volumes of common spots (up or down-regulation). Differential spots were identified by peptide fingerprinting MALDI-TOF mass spectrometry. A total of 48 identified spots (19 for leaves and 29 for roots) were grouped into protein functional classes. For leaves, 33%, 22% and 11% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively. For roots, 17.2%, 24.1% and 10.3% of the identified proteins were categorized as pertaining to the groups: metabolism, defense response and oxidative stress response, respectively.

Evaluation of glutathione S-transferase GSTM1 and GSTT1 deletion polymorphisms on type-2 diabetes mellitus risk.

BACKGROUND: Due to the activity of GSTs in the detoxification of oxidative stress products, deletion polymorphisms of GSTM1 and GSTT1 may contribute to susceptibility to T2DM, since B-cells express very low levels of antioxidant enzymes. Recently, some studies have shown an association between GSTM1-null/GSTT1-null genotypes and an increased susceptibility to T2DM. A relationship between these polymorphisms and changes in the clinical parameters of diabetic patients has also been investigated. However, the results diverge considerably among the studies. Thus, this case-control study was designed to contribute to existing knowledge, as there are no studies on this issue performed in the Brazilian population. METHODS AND FINDINGS: A total of 120 patients and 147 healthy individuals were included in this study. GSTT1 and GSTM1 deletion polymorphisms were genotyped by multiplex SYBR Green Real-Time PCR. The GSTT1-null genotype conferred a 3.2-fold increased risk to T2DM relative to the present genotype. There was no association between GSTM1-null and T2DM risk. In diabetic patients, GSTT1-null conferred higher levels of triglycerides and VLDL-cholesterol, while GSTM1-null was associated with increased levels of fasting blood glucose, glycated hemoglobin and blood pressure. We emphasized a necessity for applying log-linear analysis in order to explore an interaction between these polymorphisms properly. CONCLUSION: These results suggest that the GSTT1 polymorphism may play an important role in the pathogenesis of T2DM in the Brazilian population. This gene could then be added to a set of genetic markers to identify individuals with an increased risk for developing T2DM and complications associated with dyslipidemia in diabetic patients. Although there was no association of GSTM1 deletion polymorphism with susceptibility to T2DM, the influence of this polymorphism on important clinical parameters related to glycemia and blood pressure levels was verified. This finding suggests that both GSTM1-null and GSTT1-null may contribute to the clinical course of T2DM patients.

Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads.

In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na2SO4. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na2SO4 was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO-TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO-TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO-TCWDE retained 100% activity after 3h incubation at 55 °C. TCNSO-TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 µg of N-acetylglucosamine (NAG) maintaining 83% of initial activity.

Biodegradable and bioactive CGP/PVA film for fungal growth inhibition.

In this study, chitinolytic enzymes produced by Trichoderma asperellum were immobilized on a biodegradable film manufactured with a blend of cashew gum polysaccharide (CGP) and polyvinyl alcohol (PVA), and tested as a fungal growth inhibitor. The film was produced by casting a blend of CGP and PVA solution on glass molds. The CGP/PVA film showed 68% water solubility, tensile strength of 23.7 MPa, 187.2% elongation and 52% of mass loss after 90 days in soil. The presence of T-CWD enzymes immobilized by adsorption or covalent attachment resulted in effective inhibition of fungal growth. Sclerotinia sclerotiorum was the most sensitive organism, followed by Aspergillus niger and Penicillium sp. SEM micrograph showed that the presence of immobilized T-CWD enzymes on CGP/PVA film produced morphological modifications on vegetative and germinative structures of the microorganisms, particularly hyphae disruption and changes of spores shape.

Potential fungal inhibition by immobilized hydrolytic enzymes from Trichoderma asperellum.

The use of cell wall degrading enzymes from Trichoderma asperellum immobilized on biodegradable support is an alternative for food packaging. In this study, hydrolytic enzymes produced by T. asperellum were tested as a fungal growth inhibitor, in free form or immobilized on a biodegradable film composed of cassava starch and poly(butylene adipate-co-terephtalate) (PBAT). The inhibitory activity was tested against Aspergillus niger , Penicillium sp., and Sclerotinia sclerotiorum , microorganisms that frequently degrade food packaging. The use of chitin as carbon source in liquid medium induced T. asperellun to produce N-acetylglucosaminidase, ß-1,3-glucanase, chitinase, and protease. The presence of T. asperellun cell wall degradating enzymes (T-CWD) immobilized by adsorption or covalent attachment resulted in effective inhibition of fungal growth. The enzymatic activity of T-CWD was stronger on S. sclerotiorum than on the Aspergillus or Penicillum isolates tested. These results suggest that T-CWD can be used in a free or immobilized form to suppress fungi that degrade food packaging.

Purification and characterization of an acid phosphatase from Trichoderma harzianum.

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55 degrees C. The enzyme retained 60% of its activity after incubation at 55 degrees C for 60 min. The K (m) and V (max) values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min(-1), respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.

A hyaluronidase from Potamotrygon motoro (freshwater stingrays) venom: isolation and characterization.

Freshwater stingrays (Potamotrygon motoro) are known to cause human accidents through a sting located in its tail. In the State of Goiás, this accident happens especially during the fishing season of the Araguaia River. The P. motoro venom extracted from the sting presented hyaluronidase activity. The enzyme was purified by gel filtration on Sephacryl S-100 and ion-exchange chromatography on SP-Sepharose. A typical procedure provided 376.4-fold purification with a 2.94% yield. The molecular weight of the purified enzyme was 79 kDa as estimated by gel filtration on Sephacryl S-100. The K(m) and V(max) values for hyaluronidase, using hyaluronic acid as substrate, were 4.91 microg/ml and 2.02 U/min, respectively. The pH optimum for the enzyme was pH 4.2 and maximum activity was obtained at 40 degrees C. The hyaluronidase from P. motoro was shown to be heat instable, being stabilized by bovine albumin and DTT, and inhibited by Fe(2+), Mn(2+), Cu(2+) and heparin.

Cloning, molecular characterization and heterologous expression of AMY1, an alpha-amylase gene from Cryptococcus flavus.

A Cryptococcus flavus gene (AMY1) encoding an extracellular alpha-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) (144)DVVVNH(149), (II) (235)GLRIDSLQQ(243), (III) (263)GEVFN(267), (IV) (327)FLENQD(332), placed the enzyme in the GH13 alpha-amylase family. Furthermore, sequence comparison suggests that the C. flavusalpha-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL(-1)) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme (c. 67 kDa), part of which was due to N-glycosylation.

Chitinase from Paracoccidioides brasiliensis: molecular cloning, structural, phylogenetic, expression and activity analysis.

A full-length cDNA encoding a chitinase (Pbcts1) was cloned by screening a cDNA library from the yeast cells of Paracoccidioides brasiliensis. The cDNA consists of 1888 bp and encodes an ORF of 1218 bp corresponding to a protein of 45 kDa with 406 amino acid residues. The deduced PbCTS1 is composed of two signature family 18 catalytic domains and seems to belong to fungal/bacterial class. Phylogenetic analysis of PbCTS1 and other chitinases suggests the existence of paralogs of several chitinases to be grouped based on specialized functions, which may reflect the multiple and diverse roles played by fungi chitinases. Glycosyl hydrolase activity assays demonstrated that P. brasiliensis is able to produce and secrete these enzymes mainly during transition from yeast to mycelium. The fungus should be able to use chitin as a carbon source. The presence of an endocytic signal in the deduced protein suggests that it could be secreted by a vesicular nonclassical export pathway. The Pbcts1 expression in mycelium, yeast, during differentiation from mycelium to yeast and in yeast cells obtained from infected mice suggests the relevance of this molecule in P. brasiliensis electing PbCTS1 as an attractive drug target.

Regulation of N-acetyl-beta-D-glucosaminidase produced by Trichoderma harzianum: evidence that cAMP controls its expression.

Trichoderma harzianum is a filamentous fungus reported to be a producer of extracellular N-acetyl-beta-D-glucosaminidase (NAGase) when grown in chitin-containing medium. An approximately 64-kDa protein with NAGase activity was purified by gel filtration and ion exchange chromatography. The involvement of cyclic AMP (cAMP) in the synthesis of NAGase from T. harzianum in chitin-containing medium was also investigated. Molecules that increase the intracellular levels of cAMP, including caffeine, aluminium tetrafluoride and dinitrophenol, were used. Western blot analysis showed that NAGase synthesis was repressed by increasing the levels of intracellular cAMP. Using specific nag primers in a reverse transcription-polymerase chain reaction-based approach, NAGase synthesis was shown to be regulated at the level of gene transcription.

Molecular cloning and characterization of a cDNA encoding the N-acetyl-beta-D-glucosaminidase homologue of Paracoccidioides brasiliensis.

A cDNA encoding the N-acetyl-beta-D-glucosaminidase (NAG) protein of Paracoccidioides brasiliensis, Pb NAG1, was cloned and characterized. The 2663-nucleotide sequence of the cDNA consisted of a single open reading frame encoding a protein with a predicted molecular mass of 64.73 kDa and an isoeletric point of 6.35. The predicted protein includes a putative 30-amino-acid signal peptide. The protein as a whole shares considerable sequence similarity with 'classic' NAG. The primary sequence of Pb NAG1 was used to infer phylogenetic relationships. The amino acid sequence of Pb NAG1 has 45, 31 and 30% identity, respectively, with homologous sequences from Trichoderma harzianum, Aspergillus nidulans and Candida albicans. In particular, striking homology was observed with the active site regions of the glycosyl hydrolase group of proteins (family 20). The expected active site consensus motif G X D E and catalytic Asp and Glu residues at positions 373 and 374 were found, reinforcing that Pb NAG1 belongs to glycosyl hydrolase family 20. The nucleotide sequence of Pb nag1 and its flanking regions have been deposited, along with the amino acid sequence of the deduced protein, in GenBank under accession number AF419158.

Biochemical characterization of alpha-amylase from the yeast Cryptococcus flavus.

During our screening of amylolytic microorganisms from Brazilian fruits, we isolated a yeast strain classified as Cryptococcus flavus. When grown on starch-containing medium this strain exhibited the highest amylase production after 24 h of cultivation. The extracellular amylase from C. flavus was purified from the culture broth by a single step using chromatography on a Sephacryl S-100 column. The enzyme was purified 16.14-fold with a yield of 50.21% of the total activity. The purified enzyme was a glycoprotein with an apparent molecular mass of 75 and 84.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The enzyme lost approximately 50% of the molecular mass after treatment with glycosidases. The major end products of starch, amylose, amylopectin, pullulan and glycogen were maltose and maltotriose. The K(m) value for the pure enzyme was 0.056 mg ml(-1) with soluble starch as the substrate. Enzyme activity was optimal at pH 5.5 and 50 degrees C. The enzyme retained 90% of the activity after incubation at 50 degrees C for 60 min and was inhibited by Cu(2+), Fe(2+) and Hg(2+).

Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum.

Trichoderma asperellum produces at least two extracellular beta-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A beta-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-beta-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS-electrophoresis slab gel. The enzyme was only active toward glucans containing beta-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The K(m) and V(max) values for exo-beta-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml(-1) and 0.246 U min(-1), respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55 degrees C. Hg(2+) strongly inhibited the purified enzyme.

Involvement of g proteins and camp in the production of chitinolytic enzymes by Trichoderma harzianum

The effect of G protein modulators and cyclic AMP (cAMP) on N-acetylglucosaminidase (NAGase) production was investigated during 84 h of growth of a Trichoderma harzianum strain in chitin-containing medium. Caffeine (5 mM), N6¾2'-O-dibutyryladenosine 3'5'-cyclic monophosphate sodium salt (dBcAMP) (1 mM) and 3-isobutyl-1-methylxanthine (IBMX) (2 mM) decreased extracellular NAGase activity by 80(per cent), 77(per cent) and 37(per cent), respectively. AlCl3/KF (100 µM/10 mM and 200 µM/ 20 mM) decreased the activity by 85(per cent)and 95(per cent), respectively. Cholera (10 µ/mL) and pertussis (20 µ/mL) toxins also affected NAGase activity, causing a decrease of approximately 75(per cent). Upon all treatments, protein bands of approximately 73 kDa, 68 kDa and 45 kDa had their signals diminished whilst a 50 kDa band was enhanced only by treatment with cholera and pertussis toxins. N-terminal sequencing analysis identified the 73 kDa and 68 kDa proteins as being T. harzianum NAGase in two different truncated forms whereas the 45 kDa band comprised a T. harzianum endochitinase. The 50 kDa protein showed sequence similarity to Coriolus vesicolor cellobiohydrolase. The above results suggest that a signaling pathway comprising G-proteins, adenylate cyclase and cAMP may be involved in the synthesis of T. harzianum chitinases.