Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans

Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.UNIUBECNPqCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

A carbohydrate pulse experiment to demonstrate the sugar metabolization by S. mutans

Streptococcus mutans is a fast growing organism, of low cost and easily prepared culture medium. It has been  related  primarily to  an  elevated risk  of dental cavity development  in the host due  to the  acid-induced tooth demineralization. To prevent this disease, addition of fluoride can be required, promoting the mouth  hygiene. The  main  objective  of  this  experiment  is  to  show  the  influence  of  the  carbon  source  and fluoride on the acidogenic capacity of S.  mutans. The strain was cultivated in microaerophilia, at 37ºC for 12  hours  in  complete  medium  (stationary  phase).  The  cells  were  harvested  by  centrifugation  at  room temperature,  washed  with  saline  solution  and  suspended  in  the  same  solution.  The  absorbance  was adjusted  to  1  and  the  pH  to  7.3  using  0,1  mol/L  KOH  solution.  To  10  mL  of  the  cell  suspension,  distinct carbohydrates  (glucose,  xilose,  sucrose,  fructose  or  maltose)  were  added,  enough  to  establish  a  50 mMol/L final concentration. Fluoride was added (1 mmol/L final concentration) and the pH was monitored during  2 hours. In this  incubation  period,  the  suspension  was  kept  at  room  temperature  with  slow  stirring and  the  pH  was  monitored  each  7  minutes.  In  the  20  initial  minutes  of  incubation  with  glucose,  fructose, maltose  and  sucrose,  an  intense  and  very  similar  pH  decrease  (2.5  units)  can  be  observed.  This acidification reflects both the sugar uptake and anaerobic metabolization. After this initial acid liberation, a phase of slow pH decrease is observed, continuing up to 120 minutes of incubation. In presence of xilose, the  acidification  is  less  intense  and  reaches  a  similar  value  to  that  of  the  control  without  carbohydrate addition (decreasing  1.4 units  of pH). The  initial  acidification  in the presence of xilose  may  occur  due  to the mechanism of sugar uptake by this organism, which involves the antiport with H+. In media without the addition  of  carbohydrate,  the  acidification  may  be  due  to  the  metabolization  of  intracellular  reserves  of sugars. Fluoride affects negatively the acidogenic capacity of S. mutans for all metabolized sugars

Caracterização do processo de rigor mortis do músculo Ilio-ischiocaudalis de jacaré-do-pantanal (Caiman crocodilus yacare) e maciez da carne Characterization of rigor mortis process of muscle Ilio-ischiocaudalis of pantanal alligator (Caiman crocodilus yacare) and meat tenderness

Este trabalho utilizou seis carcaças de jacaré-do-pantanal (Caiman crocodilus yacare) com o objetivo de caracterizar o processo de rigor mortis do músculo Ílio-ischiocaudalis durante o resfriamento industrial e avaliar a maciez dessa carne. Os jacarés foram escolhidos aleatoriamente e abatidos na Cooperativa de Criadores do Jacaré do Pantanal (COOCRIJAPAN), Cáceres, Mato Grosso. Após a sangria, aferiu-se as variações das temperaturas da câmara de resfriamento, das carcaças e o pH. Foram colhidas amostras para determinação do comprimento de sarcômero, da força de cisalhamento e perdas por cozimento em diferentes intervalos de tempo (0,5, 3, 5, 7, 10, 12, 15, 24 e 36h). A temperatura da câmara de resfriamento variou de 2,6&deg;C (0,5h) a 0,9&deg;C (36h) e a temperatura média das carcaças variou de 21,0&deg;C a 4,2&deg;C, respectivamente. O pH médio inicial do músculo foi de 6,7 e o final 5,6 e a contração máxima do sarcômero do músculo Ilio-ischiocaudalis ocorreu na 15ª hora após a sangria (1,5µm). Essa carne apresentou força de cisalhamento menor que 6,0kg.<br>This paper studied six pantanal alligators (Caiman crocodilus yacare) carcass with goal of rigor mortis process characterization of Ilio-ischiocaudalis muscle during industrial cooling and meat tenderness. The alligators were randomly assembled and slaughtered at Cooperativa de Criadores do Jacaré do Pantanal (COOCRIJAPAN) - Cáceres- Mato Grosso After exsanguination, were mensured temperature of chilling room and carcasses, pH and samples were collected for determination the sarcomere length, shear force and cooking loss at different times (0.5, 3, 5, 7, 10, 12, 15, 24 and 36 hours). The temperature of chilling room varied from 2.6&deg;C (0.5h) to 0.9&deg;C (36h) and the mean carcass temperature from 21.0&deg;C to 4.2&deg;C, respectively. The mean initial pH of the muscle was 6.7 and the final was 5.6. The smallest sarcomere size ocurred at 15 hours after exsanguination (1.5µm). This meat presents shear force lower than 6.0kg