Semi-selective medium for Fusarium graminearum detection in seed samples

Fungi of the genus Fusarium cause a variety of difficult to control diseases in different crops, including winter cereals and maize. Among the species of this genus Fusarium graminearum deserves attention. The aim of this work was to develop a semi-selective medium to study this fungus. In several experiments, substrates for fungal growth were tested, including fungicides and antibiotics such as iprodiona, nystatin and triadimenol, and the antibacterial agents streptomycin and neomycin sulfate. Five seed samples of wheat, barley, oat, black beans and soybeans for F. graminearum detection by using the media Nash and Snyder agar (NSA), Segalin & Reis agar (SRA) and one-quarter dextrose agar (1/4PDA; potato 50g; dextrose 5g and agar 20g), either unsupplemented or supplemented with various concentrations of the antimicrobial agents cited above. The selected components and concentrations (g.L-1) of the proposed medium, Segalin & Reis agar (SRA-FG), were: iprodiona 0.05; nystatin 0,025; triadimenol 0.015; neomycin sulfate 0.05; and streptomycin sulfate, 0.3 added of ¼ potato sucrose agar. In the isolation from seeds of cited plant species, the sensitivity of this medium was similar to that of NSA but with de advantage of maintaining the colony morphological aspects similar to those observed in potato-dextrose-agar medium.

Ramularia areola sporulation potential in Brazilian cotton

Ramularia blight, caused by Ramularia areola, is one of the most important diseases affecting cotton crop in Brazil. For its effective control, 5-9 fungicide applications on susceptible cultivars are necessary. The aim of the present study was to evaluate, in vitro and in vivo, the sporulation potential of R. areolaisolates from different Brazilian regions at distinct temperatures. Spore production was assessed in the laboratory and under green house conditions by using leaves from plants of eight cotton cultivars. The in vitro results indicated that the potential of spore production was dependent on temperature. Maximum sporulation of the fungus occurred at 17°C for isolates from São Paulo State and 23°C for isolates from Goiás and Mato Grosso States. In the in vivo study, there was a variation in spore production according to the cultivar and the isolate. Most isolates showed to be highly aggressive on cultivars FM966 LL and DELTAOPAL. The obtained results suggest a more rational use of fungicides and cultivars with decreased fungal sporulation and can form the basis for further studies of the pathogenic variability of this fungus in cotton crops in Brazil. This is the first report on the sporulation potential of Brazilian R. areola isolates.

In vitro EVALUATION OF EUCALYPTUS ECTOMYCORRHIZAE ON SUBSTRATE WITH PHOSPHORUS DOSES FOR FUNGAL PRE-SELECTION

The benefit promoted by ectomycorrhizal depends on the interaction between symbionts and phosphorus (P) contents. Phosphorus effect on ectomycorrhizal formation and the effectiveness of these in promoting plant growth for fungal pre-selection were assessed under in vitro conditions. For P effect evaluation, Eucalyptus urophylla seedlings inoculated with four Pisolithus sp. isolates and others non-inoculated were grown on substrate containing 0.87, 1.16 and 1.72 mg P per plant. For evaluation of effectiveness and fungal pre-selection, other 30 isolates of Pisolithus sp., Pisolithus microcarpus ITA06 isolate, Amanita muscaria AM16 isolate, Scleroderma areolatum SC129 isolate were studied. D26 isolate promoted the highest plant heights for the three P doses, D51 at the lower dose and D72 at the intermediate dose. P doses did not influenced shoot fresh weight and fungal colonization. In the pre-selection of fungi, 14 isolates of Pisolithus sp., P. microcarpus ITA06 isolate and S. areolatum SC129isolate increased plant height and fresh weight. D82 isolate of Pisolithus sp. had effect singly on plant height while D17 and D58 on fresh weight. Of these, only D15, D17, D58 and ITA06 had typical ectomycorrhizae. The cultivation in vitro has shown adequate for pre-selection of ectomycorrhizal fungi. Colonization and benefits depend on species and isolate. D15, D17 and D58 of Pisolithus sp. and P. microcarpus isolate ITA06 are the most promising for nursery studies.

New perspectives on the melanocortins and their cardiovascular effects: Potential implications for the treatment of cardiovascular diseases with melanocortin analogues

The melanocortin peptides, including melanocyte-stimulating hormones, α-, β- and γ-MSH, are derived from the precursor peptide proopiomelanocortin and mediate their biological actions via five different melanocortin receptors, named from MC1 to MC5. Melanocortins have been implicated in the central regulation of energy balance and cardiovascular functions, but their local effects, via yet unidentified sites of action, in the vasculature, and their therapeutic potential in major vascular pathologies remain unclear. Therefore, the main aim of this thesis was to characterise the role of melanocortins in circulatory regulation, and to investigate whether targeting of the melanocortin system by pharmacological means could translate into therapeutic benefits in the treatment of cardiovascular diseases such as hypertension. In experiments designed to elucidate the local effects of α-MSH on vascular tone, it was found that α-MSH improved blood vessel relaxation via a nitric oxide (NO)-dependent mechanism without directly contracting or relaxing blood vessels. Furthermore, α-MSH was shown to regulate the expression and function of endothelial NO synthase in cultured human endothelial cells via melanocortin 1 receptors. In keeping with the vascular protective role, pharmacological treatment of mice with α-MSH analogues displayed therapeutic efficacy in conditions associated with vascular dysfunction such as obesity. Furthermore, α-MSH analogues elicited marked diuretic and natriuretic responses, which together with their vascular effects, seemed to provide protection against sodium retention and blood pressure elevation in experimental models of hypertension. In conclusion, the present results identify novel effects for melanocortins in the local control of vascular function, pointing to the potential future use of melanocortin analogues in the treatment of cardiovascular pathologies.

Main diseases of pejerrey (Odontesthes bonariensis) in central Argentina

Argentina's central region includes an important area covered by shallow pampean lakes and dams. In these environments, fishing of pejerrey Odontesthes bonariensis Valenciennes, 1835 (Pisces, Atherinopsidae), the most important fresh-water fish of the country, is a relevant social activity and also a considerable economic resource. The main diseases found in this species were studied from 1992 to 2003 in the provinces of Córdoba, La Rioja and Santa Fe (30º and 35º S, 61º and 67º W). Most cases were registered in high temperature months. Lernaea sp and Aeromonas hydrophila were the etiological agents most frequently found. The trophic characteristics of the aquatic environments enhanced disease processes and caused massive death of O. bonariensis, due to complex hydrochemical interactions.

Foot rot and other foot diseases of goat and sheep in the semiarid region of northeastern Brazil

This paper reports the occurrence and epidemiology of outbreaks of foot rot and other foot diseases in goats and sheep in the semiarid region of Paraíba, northeastern Brazil. Four farms were inspected for the presence of foot lesion in sheep and goats and for environmental conditions, general hygiene, pastures, and disease control measures. The prevalence of foot lesions was 19.41% (170/876) in sheep and 17.99% (52/289) in goats, ranging between 5.77% and 33.85% in different farms. Foot rot was the most common disease, affecting 12.1% of the animals examined (141/1165), but with significantly higher (p<0.05) prevalence in sheep (13.69%) than in goats (7.27%). The frequency of malignant foot rot was also significantly lower (p<0.05) in goats (9.53%) than in the sheep (40.83%). On one farm, Dorper sheep showed significantly higher (p<0.05) prevalence of foot rot (17.5%) than Santa Inês sheep (6.79%), and the number of digits affected was also higher in the former. Dichelobacter nodosus and Fusobacterium necrophorum were isolated from cases of foot rot. White line disease was found in 3.95% of the animals, sole ulcers in 1.29%, foot abscess in 1.03% and hoof overgrowth in 0.5%. The high rainfall at the time of occurrence, grazing in wetlands, clay soils with poor drainage, presence of numerous stony grounds, closure of the flocks in pens at night, and introduction of affected animals were considered predisposing factors for the occurrence of foot diseases.

Markers of Bone Turnover In Preclinical Development of Drugs for Skeletal Diseases

Skeletal tissue is constantly remodeled in a process where osteoclasts resorb old bone and osteoblasts form new bone. Balance in bone remodeling is related to age, gender and genetic factors, but also many skeletal diseases, such as osteoporosis and cancer-induced bone metastasis, cause imbalance in bone turnover and lead to decreased bone mass and increased fracture risk. Biochemical markers of bone turnover are surrogates for bone metabolism and may be used as indicators of the balance between bone resorption and formation. They are released during the remodeling process and can be conveniently and reliably measured from blood or urine by immunoassays. Most commonly used bone formation markers include N-terminal propeptides of type I collagen (PINP) and osteocalcin, whereas tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and C-terminal cross-linked telopeptide of type I collagen (CTX) are common resorption markers. Of these, PINP has been, until recently, the only marker not commercially available for preclinical use. To date, widespread use of bone markers is still limited due to their unclear biological significance, variability, and insufficient evidence of their prognostic value to reflect long term changes. In this study, the feasibility of bone markers as predictors of drug efficacy in preclinical osteoporosis models was elucidated. A non-radioactive PINP immunoassay for preclinical use was characterized and validated. The levels of PINP, N-terminal mid-fragment of osteocalcin, TRACP 5b and CTX were studied in preclinical osteoporosis models and the results were compared with the results obtained by traditional analysis methods such as histology, densitometry and microscopy. Changes in all bone markers at early timepoints correlated strongly with the changes observed in bone mass and bone quality parameters at the end of the study. TRACP 5b correlated strongly with the osteoclast number and CTX correlated with the osteoclast activity in both in vitro and in vivo studies. The concept “resorption index” was applied to the relation of CTX/TRACP 5b to describe the mean osteoclast activity. The index showed more substantial changes than either of the markers alone in the preclinical osteoporosis models used in this study. PINP was strongly associated with bone formation whereas osteocalcin was associated with both bone formation and resorption. These results provide novel insight into the feasibility of PINP, osteocalcin, TRACP 5b and CTX as predictors of drug efficacy in preclinical osteoporosis models. The results support clinical findings which indicate that short-term changes of these markers reflect long-term responses in bone mass and quality. Furthermore, this information may be useful when considering cost-efficient and clinically predictive drug screening and development assays for mining new drug candidates for skeletal diseases.

Fungal succession on leaves of Alchornea triplinervia (Spreng.) Muell. Arg. submerged in a stream of an Atlantic Rainforest in the state of São Paulo, Brazil

Leaves of Alchornea triplinervia (Spreng.) Muell. Arg. were submerged in a stream in an Atlantic Rainforest in São Paulo state, Brazil, from July/1988 to June/1989 and from July/1989 to May/1990. Fungi were isolated by the leaf disks washing technique followed by plating on culture media and also by using baiting techniques (using substrates with chitin, keratin and cellulose), what resulted on 565 fungal registers corresponding to 81 taxa. The most common species found during this study of the fungal succession were Trichoderma viride Pers. ex S.F. Gray and Fusarium oxysporum Schlecht emend. Snyd. & Hans. (23 registers), Penicillium hirsutum Dierckx (21 registers), Fusarium solani (Mart.) Appel & Wollenw. emend. Snyd. & Hans. (17), followed by 14 registers of: Cylindrocladium scoparium Morgan, Triscelophorus monosporus Ingold and Polychytrium aggregatum Ajello. Although the monthly obtained mycota had been composed by species of different taxonomic groups, the fungal succession was defined by the initial presence of typical terrestrial leaf inhabiting fungi (mostly Deuteromycotina), followed by species of Mastigomycotina and Zygomycotina. Combining culture methods and baiting techniques, it was possible to verify the presence of terrestrial fungi on the decomposition of submerged leaves and the importance of zoosporic fungi in the fungal succession. This is the first paper about the fungal succession on the decomposition of leaves submerged in a lotic ecosystem in Brazil.

Human DNA repair diseases: From genome instability to cancer

Several human genetic syndromes have long been recognized to be defective in DNA repair mechanisms. This was first discovered by Cleaver (1968), who showed that cells from patients with xeroderma pigmentosum (XP) were defective for the ability to remove ultraviolet (UV)-induced lesions from their genome. Since then, new discoveries have promoted DNA repair studies to one of the most exciting areas of molecular biology. The present work intends to give a brief summary of the main known human genetic diseases related to DNA repair and how they may be linked to acquired diseases such as cancer

Glycosuria in glomerular diseases: histopathology and clinical correlations

There are doubts about the presence of glycosuria and the progress of glomerular disease. Some reports suggest that glycosuria could be an index of a more severe tubulointerstitial lesion. We investigated the presence of glycosuria in 60 patients with primary glomerular diseases: 17 patients (28%) had glycosuria and 43 patients (72%) were glycosuria free. The two groups were similar in age, arterial pressure and sex. Serum creatinine was higher in patients with glycosuria (2.0 ± 1.7 vs 1.3 ± 0.9 mg/dl, P<0.05). The protein excretion rate was 7.5 ± 3.7 vs 5.3 ± 4.2 g/day (P>0.05) in patients with and without glycosuria, respectively, while serum albumin was lower in patients with glycosuria (1.7 ± 0.6 vs 2.7 ± 1.0 g/dl, P<0.05). Several histological forms were present in the group with glycosuria, with membranous glomerulonephritis being the most frequent. Histological evidence of tubular atrophy and interstitial fibrosis prevailed in patients with glycosuria, suggesting a poor prognosis for these patients. We may conclude that the presence of glycosuria in patients with glomerular disease is associated with more pronounced tubular atrophy and interstitial fibrosis and therefore imply a poorer prognosis.

DNA vaccines for viral diseases

DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL) responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

Apoptosis in parasites and parasite-induced apoptosis in the host immune system: a new approach to parasitic diseases

Apoptosis, a form of programmed cell death (PCD), has been described as essential for normal organogenesis and tissue development, as well as for the proper function of cell-renewal systems in adult organisms. Apoptosis is also pivotal in the pathogenesis of several different diseases. In this paper we discuss, from two different points of view, the role of apoptosis in parasitic diseases. The description of apoptotic death in three different species of heteroxenic trypanosomatids is reviewed, and considerations on the phylogenesis of apoptosis and on the eventual role of PCD on their mechanism of pathogenesis are made. From a different perspective, an increasing body of evidence is making clear that regulation of host cell apoptosis is an important factor on the definition of a host-pathogen interaction. As an example, the molecular mechanisms by which Trypanosoma cruzi is able to induce apoptosis in immunocompetent cells, in a murine model of Chagas' disease, and the consequences of this phenomenon on the outcome of the experimental disease are discussed.

Reactivity of anti-thyroid antibodies to thyroglobulin tryptic fragments: comparison of autoimmune and non-autoimmune thyroid diseases

Studies concerning the antigenicity of thyroglobulin fragments allow the characterization of the epitopes but do not consider the role of heavier antigenic fragments that could result in vivo from the action of endoproteases. Here we assess the relative importance of the fragments obtained from thyroglobulin by limited proteolysis with trypsin and compare by immunoblotting their reactivity to serum from patients with autoimmune (Graves' disease and Hashimoto's thyroiditis) and non-autoimmune (subacute thyroiditis) disease. The results showed no difference in frequency of recognition of any peptide by sera from patients with autoimmune thyroiditis. In contrast, sera from patients with subacute thyroiditis reacted more frequently with a peptide of 80 kDa. These results suggest the presence of antibody subpopulations directed at fragments produced in vivo by enzymatic cleavage of thyroglobulin. This fragment and antibodies to it may represent markers for subacute thyroiditis.

Detection of specific IgE antibodies in parasite diseases

Activation of Th1 or Th2 cells is associated with production of specific immunoglobulin isotypes, offering the opportunity to use antibody measurement for evaluation of T cell function. Schistosomiasis and visceral leishmaniasis are diseases associated with Th2 activation. However, an IgE response is not always detected in these patients. In the present study we evaluated specific IgE antibodies to S. mansoni and L. chagasi antigens by ELISA after depletion of serum IgG with protein G immobilized on Sepharose beads or RF-absorbent (purified sheep IgG antibodies anti-human IgG). In schistosomiasis patients, specific IgE to SWAP antigen was demonstrable in only 10 of 21 patients (48%) (mean absorbance ± SD = 0.102 ± 0.195) when unabsorbed serum was used. Depletion of IgG with protein G increased the number of specific IgE-positive tests to 13 (62%) and the use of RF-absorbent increased the number of positive results to 20 (95%) (mean absorbances ± SD = 0.303 ± 0.455 and 0.374 ± 0.477, respectively). Specific IgE anti-L. chagasi antibodies were not detected in unabsorbed serum from visceral leishmaniasis patients. When IgG was depleted with protein G, IgE antibodies were detected in only 3 (11%) of 27 patients, and the use of RF-absorbent permitted the detection of this isotype in all 27 visceral leishmaniasis sera tested (mean absorbance ± SD = 0.104 ± 0.03). These data show that the presence of IgG antibodies may prevent the detection of a specific IgE response in these parasite diseases. RF-absorbent, a reagent that blocks IgG-binding sites and also removes rheumatoid factor, was more efficient than protein G for the demonstration of specific IgE antibodies.

Measurement of intestinal permeability using mannitol and lactulose in children with diarrheal diseases

The excretion ratio of lactulose/mannitol in urine has been used to assess the extension of malabsorption and impairment of intestinal permeability. The recovery of lactulose and mannitol in urine was employed to evaluate intestinal permeability in children with and without diarrhea. Lactulose and mannitol probes were measured using high-performance liquid chromatography with pulsed amperometric detection (HPLC-PAD). Two groups of solutions containing 60 µM sugars were prepared. Group I consisted of glucosamine, mannitol, melibiose and lactulose, and group II of inositol, sorbitol, glucose and lactose. In the study of intra-experiment variation, a sample of 50 µl from each group was submitted to 4 successive determinations. The recovered amounts and retention times of each sugar showed a variation <2 and 1%, respectively. The estimated recovery was >97%. In the study of inter-experiment variation, we prepared 4 independent samples from groups I and II at the following concentrations: 1.0, 0.3, 0.1, 0.03 and 0.01 mM. The amounts of the sugars recovered varied by <10%, whereas the retention times showed an average variation <1%. The linear correlation coefficients were >99%. Retention (k'), selectivity (a) and efficiency (N) were used to assess the chromatographic conditions. All three parameters were in the normal range. Children with diarrhea presented a greater lactulose/mannitol ratio compared to children without diarrhea.