Production of mycelial biomass, proteases and protease inhibitors by Ganoderma lucidum under different submerged fermentation conditions.

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.

Heart failure-induced skeletal myopathy in spontaneously hypertensive rats.

BACKGROUND: Although skeletal muscle atrophy and changes in myosin heavy chain (MyHC) isoforms have often been observed during heart failure, their pathophysiological mechanisms are not completely defined. In this study we tested the hypothesis that skeletal muscle phenotype changes are related to myogenic regulatory factors and myostatin/follistatin expression in spontaneously hypertensive rats (SHR) with heart failure. METHODS: After developing tachypnea, SHR were subjected to transthoracic echocardiogram. Pathological evidence of heart failure was assessed during euthanasia. Age-matched Wistar-Kyoto (WKY) rats were used as controls. Soleus muscle morphometry was analyzed in histological sections, and MyHC isoforms evaluated by electrophoresis. Protein levels were assessed by Western blotting. STATISTICAL ANALYSIS: Student'st test and Pearson correlation. RESULTS: All SHR presented right ventricular hypertrophy and seven had pleuropericardial effusion. Echocardiographic evaluation showed dilation in the left chambers and left ventricular hypertrophy with systolic and diastolic dysfunction in SHR. Soleus weight and fiber cross sectional areas were lower (WKY 3615 ± 412; SHR 2035 ± 224 µm(2); P<0.001), and collagen fractional volume was higher in SHR. The relative amount of type I MyHC isoform was increased in SHR. Myogenin, myostatin, and follistatin expression was lower and MRF4 levels higher in SHR. Myogenin and follistatin expression positively correlated with fiber cross sectional areas and MRF4 levels positively correlated with I MyHC isoform. CONCLUSION: Reduced myogenin and follistatin expression seems to participate in muscle atrophy while increased MRF4 protein levels can modulate myosin heavy chain isoform shift in skeletal muscle of spontaneously hypertensive rats with heart failure.

Cytological and parasitological analysis of bronchoalveolar lavage fluid for the diagnosis of Angiostrongylus vasorum infection in dogs.

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of many pulmonary diseases. The aims of this work were to perform this procedure in dogs in the acute and chronic phases of an Angiostrongylus vasorum infection for cytological analysis and to evaluate the potential of this technique as a diagnostic method for this lung-heart worm. The BAL procedure was performed through the use of an endotracheal tube on seven A. vasorum infected dogs and on five non-infected dogs lined as a control group. Sixty days post-infection (dpi) active and live larvae were retrieved from the bronchoalveolar fluid (BALF) of all infected dogs. Furthermore, in one animal it was possible to retrieve larvae in its BALF before the pre-patent period. This work reports that the A. vasorum infection resulted in an increase of relative neutrophils and eosinophils counts. In contrast, there was a significant decrease in the alveolar macrophage relative count in infected animals from 60 to 330 dpi. This study shows that the BAL is an accurate technique for the diagnosis of canine angiostrongylosis. Moreover, the technique allows us to retrieve cells and other elements that line the lung surface for cytological evaluation, which provides information about inflammatory diseases, and the diagnosis and prognosis of pulmonary parasites such as A. vasorum.

Enriquecimento protéico da palma forrageira com Saccharomyces cerevisiae para alimentação de ruminantes / Protein enrichment of cactus pear with Saccharomyces cerevisiae for ruminants feeding

Avaliou-se o processo de enriquecimento protéico da palma forrageira (Opuntia ficus-indica Mill) com levedura Sacharomyces cerevisiae em cultivo semi-sólido, visando melhorar o valor nutritivo da palma para ser utilizada na alimentação de ruminantes. A levedura foi utilizada nas concentrações de 1, 2 e 3 por cento em base úmida no substrato formado pela palma forrageira, incubada em biorreatores durante 6, 12, 24 e 36 horas de fermentação. O delineamento experimental foi inteiramente ao acaso, em arranjo de parcelas subdivididas com quatro repetições. O conteúdo de proteína bruta quando se utilizou concentração de 3 por cento de inóculo, no período de seis horas, aumentou de 4,4 por cento na forma in natura para 10,4 por cento após o processamento. Os teores protéicos na concentração de 1 por cento do inóculo foram de 6,1, 8,1, 8,1 e 9,2 por cento; na concentração de 2 por cento, 9,6, 9,7, 9,8 e 9,8 por cento e na concentração de 3 por cento, 10,4, 10,4 7,9 e 7,9 por cento, nos períodos de 6, 12, 24 e 36 horas de fermentação, respectivamente. Uma fonte alternativa para arraçoamento de ruminantes, pode ser obtida pela bioconversão da palma forrageira.

The process of protein enrichment of the forage palm (Opuntia ficus-indica Mill) using the Saccharomyces cerevisiae yeast in a semi-solid culture to improve the nutritional value of forage palm for ruminants feeding was evaluated. The yeast concentrations of 1, 2 and 3 percent (wet basis) in the forage palm substrate were used. The periods of incubation were of 6, 12, 24, and 36 hours. A complete randomized experimental design in a split plot arrangement with four replicates was used. The crude protein content increased from 4.4 percent (in natura) to 10.4 percent when 3 percent of inoculums were used and the processing period was of 6 hours. The observed protein contents for 1 percent of the inoculum, used for the fermentation periods of 6, 12, 24, and 36 hours were 6.1, 8.1, 8.1, and 9.2 percent, respectively. These values were 9.6, 9.7, 9.8, and 9.8 percent for 2 percent of the inoculum, and 10.4, 10.4, 7.9, and 7.9 percent for 3 percent of the inoculum, respectively. An alternative for ruminant feeding can be obtained by bioconversion of forage palm.

Larval output of infected and re-infected dogs with Angiostrongylus vasorum (Baillet, 1866) Kamensky, 1905.

Canine angiostrongylosis is a nematode infection in domestic dogs and wild canids. A natural infection in a domestic dog frequently leads to pneumonia, loss of physical performance, coughing, anemia, cardiac insufficiency, pulmonary fibrosis and death. The main diagnostic method is based on the finding of Angiostrongylus vasorum first-stage larvae (L1) in infected dog feces. With this objective, 11 experimentally exposed to 100 third-stage larvae (L3) per kilogram of body weight (mean = 885.45 L3/animal; S.E. = 77.7). The animals were monitored for 300 days post-single-infection (PI) and the quantity of L1 output measured. Our results showed an irregular excretion of L1 and a variation in the pre-patent period (33-76 days) and the number of L1 excreted by individual animals (1-1261 L1/g). After 300 days PI, five dogs were exposed a second time and monitored for 300 days post-re-infection (PRI) (=600 days PI). The quantity of L1 output demonstrated that double exposed dogs also presented an irregular excretion of L1 but a smaller variation in the number of L1 excreted by individual animals (4-550 L1/g).

Freqüência de helmintos em gatos de Uberlândia, Minas Gerais / Frequency of helminthes parasites in cats of Uberlândia, Minas Gerais

Necropsies in 50 cats, males and females of different ages were performed from August 2000 to December 2001. Forty five (90 percent) of them had parasites. Eleven cats (22 percent) had single infection, 34 (75.6 percent) multiple infection, and only 5 (10 percent) were free of infection. The parasitic frequencies were as follow: Platynosomum fastosum (40 percent), Ancylostoma braziliensis (38 percent), Physaloptera praeputialis (34 percent), Aelurostrongylus abstrusus (18 percent), Dipylidium caninum (14 percent), Ancylostoma caninum (14 percent), Toxocara mistax (14 percent), Toxocara canis (10 percent), Trichuris campanula (6 percent), Toxascaris leonina (4 percent), Spirometra mansonoides (4 percent), Taenia taeniaeformis (4 percent) e Trichuris vulpis (2 percent).

Ectopic location of adult worms and first-stage larvae of Angiostrongylus vasorum in an infected dog.

A mongrel dog with angiostrongylosis died suddenly and was submitted to necropsy. Post-mortem findings showed adult worms in ectopic locations. This is the first report of Angiostrongylus vasorum adult worms being found within the pericardial sac and in the lumen of the bladder of an infected dog. A total of 587 A. vasorum first-stage larvae were recovered from different organs and tissues after concentration using Baermann apparatus.