Production and partial characterization of lipase from Penicillium verrucosum obtained by submerged fermentation of conventional and industrial media

The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. Current studies on lipase production by submerged fermentation involve the use of agro-industrial residues aiming at increasing economic attractiveness. Based on these aspects, the objective of this work was to investigate lipase production by Penicillium verrucosum in submerged fermentation using a conventional medium based on peptone, yeast extract, NaCl and olive oil, and an industrial medium based on corn steep liquor, Prodex Lac (yeast hydrolysate), NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained. Kinetics of lipase production was evaluated and the highest enzymatic activities, of 3.15 and 2.22 U.mL-1, were observed when conventional and industrial media were used, respectively. The enzymatic extract showed optimal activity in the range from 30 to 40 °C and at pH 7.0. Although the industrial medium presents economical advantages over the conventional medium, the presence of agro-industrial residues rich in nitrogen and other important nutrients seemed to contribute to a reduction in lipase activity.

Comparison of different methods for the determination of total organic carbon and humic substances in Brazilian soils

ABSTRACTAiming to compare three different methods for the determination of organic carbon (OC) in the soil and fractions of humic substances, seventeen Brazilian soil samples of different classes and textures were evaluated. Amounts of OC in the soil samples and the humic fractions were measured by the dichromate-oxidation method, with and without external heating in a digestion block at 130 °C for 30 min; by the loss-on-ignition method at 450 °C during 5 h and at 600 °C during 6 h; and by the dry combustion method. Dry combustion was used as reference in order to measure the efficiency of the other methods. Soil OC measured by the dichromate-oxidation method with external heating had the highest efficiency and the best results comparing to the reference method. When external heating was not used, the mean recovery efficiency dropped to 71%. The amount of OC was overestimated by the loss-on-ignition methods. Regression equations obtained between total OC contents of the reference method and those of the other methods showed relatively good adjustment, but all intercepts were different from zero (p < 0.01), which suggests that more accuracy can be obtained using not one single correction factor, but considering also the intercept. The Walkley-Black method underestimated the OC contents of the humic fractions, which was associated with the partial oxidation of the humin fraction. Better results were obtained when external heating was used. For the organic matter fractions, the OC in the humic and fulvic acid fractions can be determined without external heating if the reference method is not available, but the humin fraction requires the external heating.

Hepatic regeneration after partial hepatectomy in mice infected with Schistosoma mansoni, at the acute and chronic phases of the disease

Outbred male albino mice normal or infected with 30 cercariae of Schistosoma mansoni (LE strain) were submitted to 65% hepatectomy during the acute (70 days) and chronic phase (160 days) phases of the disease. A group of the infected animals was treated with 400 mg/kg of oxamniquine during the acute phase before hepatectomy. Non-infected, infected and treated but not hepatectomized animals were kept as controls. Hepatic regeneration was evaluated by incorporation of tritiated thymidine, intraperitoneally injected into non-hepatectomized and hepatectomized animals, 24 hours after surgery. The results showed that removal of 65% of the hepatic parenchyma, during the acute phase, led to a statistically significant increase of thymidine incorporation, when compared with the uninfected hepatectomized controls. This phenomenon was not observed at the chronic phase. Treatment with oxamniquine administered during the acute phase led to a decrease in thymidine incorporation rate 160 days after infection (90 days after treatment) and 24 hours after hepatectomy. The data suggest that infection with S. mansoni represents a considerable stimulus for the regenerative capacity of the liver during the acute, but not the chronic phase of disease.

Partial chemical characterization of antigenic preparations of chromoblastomycosis agents

Antigenic preparations (saline, methylic, metabolic and exoantigens) of four agents of chromoblastomycosis, Fonsecaea pedrosoi, Phialophora verrucosa, Cladophialophora (Cladosporium) carrionii and Rhinocladiella aquaspersa were obtained. Partial chemical characterization of these antigenic preparations was obtained by determination of the levels of total lipids, protein, and carbohydrates, and identification of the main sterols and carbohydrates. Methylic antigens presented the highest lipid contents, whereas metabolic antigens showed the highest carbohydrate content. Total lipid, protein, and carbohydrate levels were in the range of 2.33 to 2.00mg/ml, 0.04 to 0.02 mg/ml and 0.10 to 0.02 mg/ml, respectively, in the methylic antigens and in the range of 0.53 to 0.18mg/ml, 0.44 to 0.26mg/ml, and 1.82 to 1.02 mg/ml, respectively, in saline antigens. Total lipid, protein, and carbohydrate contents were in the range of 0.55 to 0.20mg/ml, 0.69 to 0.57mg/ml and 10.73 to 5.93mg/ml, respectively, in the metabolic antigens, and in the range of 0.55 to 0.15mg/ml, 0.62 to 0.20mg/ml and 3.55 to 0.42mg/ml, respectively, in the exoantigens. Phospholipids were not detected in the preparations. Saline and metabolic antigens and exoantigens presented hexose and the methylic antigen revealed additional pentose units in their composition. The UV light absorption spectra of the sterols revealed squalene and an ergosterol fraction in the antigens. The characterization of these antigenic preparations may be useful for serological evaluation of patients of chromoblastomycosis.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Ascaris suum: partial fractionation of metabolic antigens from "in vitro" cultured larvae

Ascaris suum metabolic antigens were obtained frorn second and early third stage larvae cultured in vitro in supplemented Eagle's minimum essential medium. Metabolic antigens harvested after 12 and 16 days from in vitro cultures were eluted through Bio-Gel Al.5. Three main elution peaks were identified, dialysed, lyophilized and injected into mice with 4% sodium alginate. Peak 11 from elution of two preparations of metabolic antigens protected mice against a chállenge infection of 10,000 A. suum embryonated eggs.

Induction of male sterility through manipulation of genetic mechanisms present in vector species of Triatominae. II. Partial restoration of male fertility

The development of integrated measures which involve sterile mate release to supplement the conventional insecticidal techniques used in controlagainst insects of medical importance, raised the question, whether the vectors of Chagas'disease possess the natural mechanisms by manipulation of which they may be controlled. Results of earlier expenments, that had been published previously, were restricted to fragmentary information that raised various questions, the answer to which became available in the study herein described. Interspecific hybrids were produced from reciprocal crosses between T. pseudomaculata and T. sórdida and from unilateral crosses between female T. pseudomaculata and male. T. infestans. These females mated with males, laid less than the normal complement of eggs, but offspring was relatively abundant. When T. pseudomaculata females were paired with T. brasiliensis males, hybridization was more difficult because few of the females mated and those that did had a strongly reduced fertility. Adults emerged from ali crosses but exhibited sex disproportion, females predominating in all populations but one. The two Rhodnius species tested were also found to cross, but only when female R. prolixus were paired with male R. neglectus. These females laid a relatively high complement o f eggs, had a strongly reduced fertility, but 50% of the fertile eggs developed into vigorous adults, males predominating females. Neither type of hybrid male elicited fertilized eggs from either parental type of female, through their vesicula seminal is were found to be packed with spermatozoa, some normal looking and moving, others underdeveloped and motionless. Although, no artificial insemination was performed, the sperm in itself did not appear to be the prime inducer of sterility. Females paired with these hybrids did mate, sperm was transfered, as evidenced by the discharged spermatophores smeared with sperm, but did notcontain spermatozoa in their spermatecae. The failure of the sperm to migrate to the spermatecae indicate prezygotic pos-copulation incompatibility, thus the hybrid male can't be used to suppress populations. The female hybrids mated with parent males of either species had reduced fertility and ther sons were sterile as were those of their fertile daughters. However, continous backcrossing of the hybrid females and their female progeny to parental males partially restored fertility of the males and increased fertility of females, as scored by egg hatchability. Fertility of hybrid females, measured by the yield of adults capable to reproduce, indicated that the reproductive perfomance decreased when hybrid females and their daughters were backcrossed additional generations to parental males. It is tentatively suggested that hybrid females could be used for suppression if they compete efficiently with wild females.

Role of partial hepatectomy on Capillaria hepatica-induced hepatic fibrosis in rats

It is known that hepatic fibrosis may regress following partial hepatectomy, since the hepatic parenchyma regenerates very rapidly, but not the excess of fibrous tissue. The present study evaluated this hypothesis by observing the behavior of systematized septal fibrosis induced by either 30 or 90-day-old Capillaria hepatica infection, in rats subjected to partial hepatectomy. The results revealed that the morphology of the fibrosis was unaffected, but its relative quantity within the microscope field appeared significantly decreased, as a consequence of the increased liver tissue mass following regeneration.

A dieta e o consumo diário de alimento de Cichla monoculus na Amazônia Central

Avaliar a dieta, o ritmo alimentar e a taxa de consumo diária de alimento dos peixes pode permitir estimar a relação entre a alimentação e o crescimento, a pressão de predação sobre espécies de presas, a limitação alimentar durante estações do ano e a competição intra e inter-específica. Estas informações são desconhecidas para C.monoculus na Amazônia Central e aqui são apresentados dados sobre a ecologia trófica desta espécie durante quatro estações hidrológicas. A área de estudo incluía três lagos de várzea na Amazônia Central, durante os períodos de agosto de 1997 a julho de 1998. Os estudos da dieta e do ritmo alimentar foram feitos através das análises dos conteúdos estomacais. A taxa de evacuação gástrica foi estimada experimentalmente. O consumo diário de alimento foi calculado a partir dos modelos de Elliot & Persson e de Eggers. A intensidade de alimentação da espécie foi baixa durante a seca. A dieta do C. monoculus foi basicamente piscívora e composta de nove famílias de peixes e uma de camarão, apresentando variações no decorrer das estações hidrológicas e com o tamanho do peixe. A taxa de evacuação gástrica foi 16,9% h-1. O consumo diário de alimento, que não foi diferente nas quatro estações hidrológicas, teve média igual a 2,23% do peso corporal. Este valor é baixo comparado a outros estudos estimados para peixes tropicais. Indicando que esta espécie come relativamente pouco.

Biomass equations for forest regrowth in the eastern Amazon using randomized branch sampling

Forest regrowth occupies an extensive and increasing area in the Amazon basin, but accurate assessment of the impact of regrowth on carbon and nutrient cycles has been hampered by a paucity of available allometric equations. We develop pooled and species-specific equations for total aboveground biomass for a study site in the eastern Amazon that had been abandoned for 15 years. Field work was conducted using randomized branch sampling, a rapid technique that has seen little use in tropical forests. High consistency of sample paths in randomized branch sampling, as measured by the standard error of individual paths (14%), suggests the method may provide substantial efficiencies when compared to traditional procedures. The best fitting equations in this study used the traditional form Y=a×DBHb, where Y is biomass, DBH is diameter at breast height, and a and b are both species-specific parameters. Species-specific equations of the form Y=a(BA×H), where Y is biomass, BA is tree basal area, H is tree height, and a is a species-specific parameter, fit almost as well. Comparison with previously published equations indicated errors from -33% to +29% would have occurred using off-site relationships. We also present equations for stemwood, twigs, and foliage as biomass components.

Clinical outcome of patients with familial hypercholesterolemia and coronary artery disease undergoing partial ileal bypass surgery

Familial hypercholesterolemia is characterized by high serum levels of total cholesterol and LDL-cholesterol. It may be homozygous or heterozygous. In homozygous patients, LDL-cholesterol levels range from 500 to 1000mg/dL and coronary artery disease is precocious, usually manifesting itself between the 2nd and 3rd decades of life. The diagnosis is often made by the presence of xanthoma tuberosum and tendinous xanthomas that appear between the 1st and 2nd decades of life. The use of high doses of statins or even unusual procedures (apheresis, partial ileal bypass surgery, liver transplantation, gene therapy), or both, is necessary for increasing survival and improving quality of life, because a reduction in cholesterol levels is essential for stabilizing the coronary artery disease and reducing xanthomas. We report our experience with 3 patients with xanthomatous familial hypercholesterolemia and coronary artery disease, who underwent partial ileal bypass surgery. Their follow-up over the years (approximately 8 years) showed a mean 30% reduction in total cholesterol, with a significant reduction in the xanthomas and stabilization of the coronary artery disease.

Surgical treatment of partial atrioventricular septal defect: functional analysis of the mitral valve in the postoperative period

OBJECTIVE: To study mitral valve function in the postoperative period after correction of the partial form of atrioventricular septal defect. METHODS: Fifty patients underwent surgical correction of the partial form of atrioventricular septal defect. Their mean age was 11.8 years and 62% of the patients were males. Preoperative echocardiography showed moderate and severe mitral insufficiency in 44% of the patients. The mitral valve cleft was sutured in 45 (90%) patients (group II - GII). Echocardiographies were performed in the early postoperative period, and 6 and 12 months after hospital discharge. RESULTS: The patients who had some type of arrhythmia in the postoperative period had ostium primum atrial septal defect of a larger size (2.74 x 2.08 cm). All 5 patients in group I (GI), who did not undergo closure of the cleft, had a competent mitral valve or mild mitral insufficiency in the preoperative period. One of these patients began to have moderate mitral insufficiency in the postoperative period. On the other hand, in GII, 88.8% and 82.2% of the patients had competent mitral valve or mild mitral insufficiency in the early and late postoperative periods, respectively. CONCLUSION: The mitral valve cleft was repaired in 90% of cases. Echocardiography revealed competent mitral valve or mild mitral insufficiency in 88.8% and 82.2% of GII patients in the early and late postoperative periods, respectively.

Sex-Specific Equations to Estimate Maximum Oxygen Uptake in Cycle Ergometry

AbstractBackground:Aerobic fitness, assessed by measuring VO2max in maximum cardiopulmonary exercise testing (CPX) or by estimating VO2max through the use of equations in exercise testing, is a predictor of mortality. However, the error resulting from this estimate in a given individual can be high, affecting clinical decisions.Objective:To determine the error of estimate of VO2max in cycle ergometry in a population attending clinical exercise testing laboratories, and to propose sex-specific equations to minimize that error.Methods:This study assessed 1715 adults (18 to 91 years, 68% men) undertaking maximum CPX in a lower limbs cycle ergometer (LLCE) with ramp protocol. The percentage error (E%) between measured VO2max and that estimated from the modified ACSM equation (Lang et al. MSSE, 1992) was calculated. Then, estimation equations were developed: 1) for all the population tested (C-GENERAL); and 2) separately by sex (C-MEN and C-WOMEN).Results:Measured VO2max was higher in men than in WOMEN: -29.4 ± 10.5 and 24.2 ± 9.2 mL.(kg.min)-1 (p < 0.01). The equations for estimating VO2max [in mL.(kg.min)-1] were: C-GENERAL = [final workload (W)/body weight (kg)] x 10.483 + 7; C-MEN = [final workload (W)/body weight (kg)] x 10.791 + 7; and C-WOMEN = [final workload (W)/body weight (kg)] x 9.820 + 7. The E% for MEN was: -3.4 ± 13.4% (modified ACSM); 1.2 ± 13.2% (C-GENERAL); and -0.9 ± 13.4% (C-MEN) (p < 0.01). For WOMEN: -14.7 ± 17.4% (modified ACSM); -6.3 ± 16.5% (C-GENERAL); and -1.7 ± 16.2% (C-WOMEN) (p < 0.01).Conclusion:The error of estimate of VO2max by use of sex-specific equations was reduced, but not eliminated, in exercise tests on LLCE.

A determinação dos números de indivíduos mínimos necessários na experimentação genética

The main object of the present paper consists in giving formulas and methods which enable us to determine the minimum number of repetitions or of individuals necessary to garantee some extent the success of an experiment. The theoretical basis of all processes consists essentially in the following. Knowing the frequency of the desired p and of the non desired ovents q we may calculate the frequency of all possi- ble combinations, to be expected in n repetitions, by expanding the binomium (p-+q)n. Determining which of these combinations we want to avoid we calculate their total frequency, selecting the value of the exponent n of the binomium in such a way that this total frequency is equal or smaller than the accepted limit of precision n/pª{ 1/n1 (q/p)n + 1/(n-1)| (q/p)n-1 + 1/ 2!(n-2)| (q/p)n-2 + 1/3(n-3) (q/p)n-3... < Plim - -(1b) There does not exist an absolute limit of precision since its value depends not only upon psychological factors in our judgement, but is at the same sime a function of the number of repetitions For this reasen y have proposed (1,56) two relative values, one equal to 1-5n as the lowest value of probability and the other equal to 1-10n as the highest value of improbability, leaving between them what may be called the "region of doubt However these formulas cannot be applied in our case since this number n is just the unknown quantity. Thus we have to use, instead of the more exact values of these two formulas, the conventional limits of P.lim equal to 0,05 (Precision 5%), equal to 0,01 (Precision 1%, and to 0,001 (Precision P, 1%). The binominal formula as explained above (cf. formula 1, pg. 85), however is of rather limited applicability owing to the excessive calculus necessary, and we have thus to procure approximations as substitutes. We may use, without loss of precision, the following approximations: a) The normal or Gaussean distribution when the expected frequency p has any value between 0,1 and 0,9, and when n is at least superior to ten. b) The Poisson distribution when the expected frequecy p is smaller than 0,1. Tables V to VII show for some special cases that these approximations are very satisfactory. The praticai solution of the following problems, stated in the introduction can now be given: A) What is the minimum number of repititions necessary in order to avoid that any one of a treatments, varieties etc. may be accidentally always the best, on the best and second best, or the first, second, and third best or finally one of the n beat treatments, varieties etc. Using the first term of the binomium, we have the following equation for n: n = log Riim / log (m:) = log Riim / log.m - log a --------------(5) B) What is the minimun number of individuals necessary in 01der that a ceratin type, expected with the frequency p, may appaer at least in one, two, three or a=m+1 individuals. 1) For p between 0,1 and 0,9 and using the Gaussean approximation we have: on - ó. p (1-p) n - a -1.m b= δ. 1-p /p e c = m/p } -------------------(7) n = b + b² + 4 c/ 2 n´ = 1/p n cor = n + n' ---------- (8) We have to use the correction n' when p has a value between 0,25 and 0,75. The greek letters delta represents in the present esse the unilateral limits of the Gaussean distribution for the three conventional limits of precision : 1,64; 2,33; and 3,09 respectively. h we are only interested in having at least one individual, and m becomes equal to zero, the formula reduces to : c= m/p o para a = 1 a = { b + b²}² = b² = δ2 1- p /p }-----------------(9) n = 1/p n (cor) = n + n´ 2) If p is smaller than 0,1 we may use table 1 in order to find the mean m of a Poisson distribution and determine. n = m: p C) Which is the minimun number of individuals necessary for distinguishing two frequencies p1 and p2? 1) When pl and p2 are values between 0,1 and 0,9 we have: n = { δ p1 ( 1-pi) + p2) / p2 (1 - p2) n= 1/p1-p2 }------------ (13) n (cor) We have again to use the unilateral limits of the Gaussean distribution. The correction n' should be used if at least one of the valors pl or p2 has a value between 0,25 and 0,75. A more complicated formula may be used in cases where whe want to increase the precision : n (p1 - p2) δ { p1 (1- p2 ) / n= m δ = δ p1 ( 1 - p1) + p2 ( 1 - p2) c= m / p1 - p2 n = { b2 + 4 4 c }2 }--------- (14) n = 1/ p1 - p2 2) When both pl and p2 are smaller than 0,1 we determine the quocient (pl-r-p2) and procure the corresponding number m2 of a Poisson distribution in table 2. The value n is found by the equation : n = mg /p2 ------------- (15) D) What is the minimun number necessary for distinguishing three or more frequencies, p2 p1 p3. If the frequecies pl p2 p3 are values between 0,1 e 0,9 we have to solve the individual equations and sue the higest value of n thus determined : n 1.2 = {δ p1 (1 - p1) / p1 - p2 }² = Fiim n 1.2 = { δ p1 ( 1 - p1) + p1 ( 1 - p1) }² } -- (16) Delta represents now the bilateral limits of the : Gaussean distrioution : 1,96-2,58-3,29. 2) No table was prepared for the relatively rare cases of a comparison of threes or more frequencies below 0,1 and in such cases extremely high numbers would be required. E) A process is given which serves to solve two problemr of informatory nature : a) if a special type appears in n individuals with a frequency p(obs), what may be the corresponding ideal value of p(esp), or; b) if we study samples of n in diviuals and expect a certain type with a frequency p(esp) what may be the extreme limits of p(obs) in individual farmlies ? I.) If we are dealing with values between 0,1 and 0,9 we may use table 3. To solve the first question we select the respective horizontal line for p(obs) and determine which column corresponds to our value of n and find the respective value of p(esp) by interpolating between columns. In order to solve the second problem we start with the respective column for p(esp) and find the horizontal line for the given value of n either diretly or by approximation and by interpolation. 2) For frequencies smaller than 0,1 we have to use table 4 and transform the fractions p(esp) and p(obs) in numbers of Poisson series by multiplication with n. Tn order to solve the first broblem, we verify in which line the lower Poisson limit is equal to m(obs) and transform the corresponding value of m into frequecy p(esp) by dividing through n. The observed frequency may thus be a chance deviate of any value between 0,0... and the values given by dividing the value of m in the table by n. In the second case we transform first the expectation p(esp) into a value of m and procure in the horizontal line, corresponding to m(esp) the extreme values om m which than must be transformed, by dividing through n into values of p(obs). F) Partial and progressive tests may be recomended in all cases where there is lack of material or where the loss of time is less importent than the cost of large scale experiments since in many cases the minimun number necessary to garantee the results within the limits of precision is rather large. One should not forget that the minimun number really represents at the same time a maximun number, necessary only if one takes into consideration essentially the disfavorable variations, but smaller numbers may frequently already satisfactory results. For instance, by definition, we know that a frequecy of p means that we expect one individual in every total o(f1-p). If there were no chance variations, this number (1- p) will be suficient. and if there were favorable variations a smaller number still may yield one individual of the desired type. r.nus trusting to luck, one may start the experiment with numbers, smaller than the minimun calculated according to the formulas given above, and increase the total untill the desired result is obtained and this may well b ebefore the "minimum number" is reached. Some concrete examples of this partial or progressive procedure are given from our genetical experiments with maize.