Direct Evidence for Intracellular Homeostasis in Mammalian Cells: Insulin-independent Glucose Metabolisms

In our previous study, carbonates, NaHCO3 and Na2CO3, influence glucose metabolism in vitro, using Py-3Y1-S2 rat fibroblast cells, and these compounds accelerate significantly glucose consumption. In the present study, the effects of the carbonates on glucose metabolism were examined to determine whether these effects are universal among different cell lines, VERO green monkey kidney cells, TE-13 human esophageal cancer cells, and HepG2 human cells. Glucose was completely converted to lactate, which disappeared gradually from the culture medium. However, the disappearance of lactate from the medium was independent of carbonates. The present study clarified that NaHCO3 and Na2CO3 directly regulate glucose metabolism among different cell lines via an insulin-independent pathway, that is, intracellular homeostasis.

Hepatoprotective and antioxidant properties of Suaeda maritima (L.) Dumort ethanolic extract on concanavalin-A induced hepatotoxicity in rats.

Hepatoprotective and antioxidant properties of Suaeda maritima (L.) Dumort on concanavalin-A induced stress in Wistar albino rats have been reported. Rats were administered with ethanolic extract of Suaeda maritima at the concentration of 75, 150 and 300 mg/kg of body wt. for 9 days and concanavalin-A was administrated (iv) 12 mg/kg on 9th day. Rats in concanavalin-A administered group showed elevated levels of AST, ALT, ALP and bilurubin. Pretreatment of rats with ethanolic extract (300 mg/kg) significantly reduced these serum parameters compared to concavalin-A administered group. Histopathological examination of liver sections showed that, normal liver architecture was disturbed by hepatotoxin intoxication. The extract treated group and silymarin treated group retained the normal cell architecture, although less visible changes were observed. Preliminary phytochemical analysis showed the presence of triterpenioids and may be responsible for the hepatoprotective activity. The LD50 was calculated as 3 g/kg of the body weight. IC50 values of hydroxyl (52.21±1.32g/ml) and nitric oxide radicals (09.14±0.94 g/ml) scavenging results showed comparable activity with vitamin-C. Results of this study may be useful for the development of herbal medicine from Suaeda maritima for the treatment of hepatitis.

Purificación de una fracción de antígenos de tripomastigotes de Trypanosoma cruzi para el diagnóstico de la enfermedad de Chagas / Purification of a trypomastigote antigen fraction from Trypanosoma cruzi for Chagas’ disease diagnosis

Los antígenos excretados/secretados por las formas tripomastigotes de T. cruzi (antígenos TESA) pertenecen a la familia de las transialidasas, las cuales son responsables de la transferencia de ácido siálico exógeno a moléculas aceptadoras en la superficie de los tripomastigotes. En el presente trabajo se purifican varias proteínas de los antígenos TESA utilizando cromatografía de afinidad con resina de sefarosa B4-concanavalina A, con la intención de ser utilizados en el diagnóstico de la enfermedad de Chagas. El buffer de elución contenía una mezcla de α-D-manopiranósido y α-D-glucopiranósido. Se realizó electroforesis unidimensional en gel con poliacrilamida para identificar las bandas purificadas y la prueba de inmunoelectrotransferencia para visualizar las bandas reactivas con el pool de sueros de individuos con infección por T. cruzi. El gel teñido con azul de Coomassie coloidal permitió visualizar 3 bandas de aproximadamente 220, 170, y 20 kDa. La inmunoelectrotransferencia utilizando un pool de sueros positivos, confirmados para la infección por T. cruzi, reveló 5 bandas inmunogénicas de 220, 120, 85, 50 y 32 kDa mientras que el revelado con diaminobenzidina permitió observar las bandas de 220, 120, 85, 50 32 y 20 kDa. Asimismo las bandas purificadas no fueron reconocidas en la inmunoelectrotransferencia por el pool de sueros confirmados como negativos. Estos resultados sugieren el potencial de estas proteínas purificadas de TESA para ser usadas como nueva herramienta para el diagnóstico de la enfermedad de Chagas.

Trypanosoma cruzi excreted/secreted antigens (TESA) belong to the transialidase family, which are responsible for the transfer of exogenous sialic acid to accepting molecules at the trypomastigote surface. In the present study we purified several proteins from TESA antigens using affinity chromatography with sepharose B4-concanavalin A resin, with the purpose of using them for Chagas’ disease diagnosis. The elution buffer contained a mixture of α-D-manopiranosid and α-D-glucopiranosid. A unidimensional electrophoresis in polyacrilamide gel to identify the purified bands, and an immunoelectrotransference test with a pool of sera from T. cruzi infected individuals to visualize the reactive bands were carried out. The colloidal Coomassie blue stained gel allowed visualizing 3 bands of approximately 220, 170 and 20 kDa. The immunoelectrotransference using a pool of positive sera with confirmed T. cruzi infection showed 5 immunogenic 220, 120, 50 and 32 kDa bands, while a developing with diaminobenzidine showed 220, 120, 85, 50, 32 and 20 kDa bands. The purified bands were not recognized in an immunoelectrotransference test when a pool of confirmed negative sera was used. These results suggest the potential of these TESA purified proteins for using them as a new tool for Chagas’ disease diagnosis.

Serological reactivity of different antigenic preparations of Leishmania (Leishmania) amazonensis and the Leishmania braziliensis complex / Reatividade sorológica frente a diferentes preparações antigênicas de Leishmania (Leishmania) amazonensis e do complexo Leishmania braziliensis

Total antigen from Leishmania (Leishmania) amazonensis and isolates from the Leishmania braziliensis complex, along with their respective antigenic fractions obtained by affinity chromatography on concanavalin-A-Sepharose and jacalin-agarose columns evaluated using immunoenzymatic ELISA assay. For this, serum samples from 229 patients were used, grouped as American tegmental leishmaniasis (nº=58), visceral leishmaniasis (nº=28), Chagas disease (nº=49), malaria (nº=32), tuberculosis (nº=13) and healthy volunteers (nº=49). Samples from American tegmentary leishmaniasis showed higher reactivity with antigens isolated from the Leishmania braziliensis complex than with antigens from Leishmania amazonensis (p<0.001). ELISA assays showed a sensitivity range from 60 percent to 95 percent with antigens isolated from the Leishmania braziliensis complex. There was marked nonspecific reactivity among serum samples with the use of antigenic fractions binding with concanavalin-A and jacalin from both Leishmania complexes, in comparison with other antigens (p<0.001). The results presented in this study suggest that the use of homologous antigens increases the efficiency of anti-Leishmania immunoglobulin detection, which may be very valuable for diagnostic purposes.

Antígeno total de Leishmania (Leishmania) amazonensis e isolado do complexo Leishmania brazilienis, assim como suas respectivas frações antigênicas obtidas por cromatografia de afinidade em coluna de concanavalina-A ligada a sepharose e Jacalina ligada a agarose foram avaliadas por ensaio imunoenzimático ELISA. Para tanto, foram utilizadas amostras de soros de 229 pacientes agrupadas em leishmaniose tegumentar americana (nº=58), leishmaniose visceral (nº=28), doença de Chagas (nº=49), malaria (nº=32), tuberculose (nº=13) e voluntários saudáveis (nº=49). Houve maior reatividade das amostras de leishmaniose tegumentar americana com a utilização dos antígenos obtidos do isolado do complexo Leishmania braziliensis quando comparado com antígenos de Leishmania amazonensis (p<0,001). Observou-se ainda que a sensibilidade do teste ELISA variou de 60 a 95 por cento entre os antígenos obtidos do isolado do complexo Leishmania braziliensis. Houve acentuada reatividade inespecífica das amostras de soros com a utilização das frações antigênicas ligantes de Concanavalina-A e Jacalina de ambos os complexos Leishmania em comparação aos demais antígenos (p<0,001). Os resultados apresentados no presente trabalho sugerem que a utilização de antígenos homólogos aumentam a eficiência de detecção de imunoglobulina anti-Leishmania o que pode ser de grande valia para o propósito de diagnóstico.

Efecto de la concanavalina a sobre la actividad de las enzimas a-amilasa pancreática y tripsina en pollos de engorde / The effect of concanavalin a on pancreatic amylase and trypsin activity in broiler chickens

Con el propósito de determinar el efecto de la Concanavalina A (Con A) sobre la actividad de las enzimas a-amilasa pancreática y tripsina en pollos de engorde de 3 y 6 semanas de edad, se realizaron dos experimentos bajo condiciones in vitro. En el primero, la actividad de la enzima a-amilasa pancreática fue determinada en muestras de mucosa duodenal, para lo cual se diseñaron 5 tratamientos: ausencia de Con A (T0), presencia de Con A (T1), Con A preincubada durante 30 minutos con la enzima (T2) o con el sustrato (T3) y Caseína (T4). En el segundo experimento se evaluó el efecto de la Con A sobre la actividad de la tripsina en homogenados de páncreas, aplicando 2 tratamientos: presencia de Con A (T0) y ausencia de Con A (T1). La Con A se utilizó a una concentración semejante a la del sustrato correspondiente para cada enzima. Los resultados fueron analizados a través del Análisis de Varianza de Kruskal-Wallis. La Con A inhibió significativamente la actividad específica de la enzima a-amilasa pancreática, tanto en la tercera como en la sexta semana de edad. No hubo diferencia en la actividad de la enzima entre semanas. La preincubación de la lectina con la enzima afectó significativamente la actividad de la a-amilasa pancreática. No hubo efecto de la preincubación de la lectina con el sustrato. La tripsina no fue inhibida por la Con A bajo las condiciones del ensayo, posiblemente asociado al efecto inhibitorio de la actividad biológica que ejerce la caseína sobre la Con A.

Two trials were conducted to assess the effect of Concanavalin A on pancreatic a-amylase and trypsin activity in broiler chickens (3 and 6 week old). Pancreatic a-amylase activity was determined in mucous duodenal, applying 5 treatments: Control (without Con A, T0), Con A (T1), Con A preincubated during 30 minutes with enzyme (T2), Con A preincubated during 30 minutes with substrate (T3) and Casein (T4). In the second experiment, the effect of Con A on trypsin activity was studied in pancreas homogenate, applying 2 treatments: Control (without Con A, T0) and Con A (T1). The Con A was used in a similar concentration to corresponding substrate for each enzyme. The results were analyzed through Kruskal-Wallis. The Con A significantly inhibited specific activity of pancreatic a-amylase during the third and sixth week of age. There was no difference in the activity of the enzyme between weeks. The preincubation of lectin with enzyme significantly affected pancreatic a-amylase. There was not effect of preincubation of lectin with substrate. Trypsin was not inhibited by Con A under experimental conditions, possibly due to inhibitory effect of biological activity that exerts the casein on Con A.

Efectos del tostado sobre el valor de energía metabolizable verdadera y el contenido de factores antinutricionales de harinas de granos / De canavalia ensiformis (l.) Effects of toasting on true metabolizable energy value and content of antinutritional factors of canavalia ensiformis (l.) Seed meals

El efecto del tostado sobre el valor de energía metabolizable verdadera corregida para balance de nitrógeno nulo (EMVn) y el contenido de factores antinutricionales (FAN) de harinas tostadas de Canavalia fue investigado en un ensayo de balance. Adicionalmente, se determinó el contenido de lisina reactiva, la solubilidad de la proteína y el color en las harinas evaluadas. Se asignaron al azar 5 gallos cecotomizados a cada harina (cruda o tostada) a las siguientes temperaturas y tiempos: 180; 200; 220; and 230°C/3 min; 230 y 240°C/2 min; 230 y 240°C/1 min. Cada gallo fue intubado con 40g de harina y las heces fueron recolectadas durante 72 horas. La Concanavalina A (Con A) en las harinas tostadas fue detectada determinando la unión de esta lectina a la mucosa duodenal por inmunohistoquímica. Ninguna de las harinas tostadas mostró actividad hemaglutinante de la Con A, pero se observó unión de esta lectina a la mucosa duodenal con una reacción inmunohistoquímica de moderada a débil. El tostado a 220; 230°C/3 min ó 240°C/2 min redujo la canavanina en más del 90% en relación con la harina cruda. No se observaron diferencias significativas (P < 0,05) entre el valor de EMVn de la harina cruda y el de las harinas tostadas a 200; 220; 230°C/3 min; 240°C/1 ó 240°C/2min mientras que, el tostado a 180°C/3min; 230°C/1min ó 230°C/2min redujo significativamente (P < 0,05) la EMVn. La lisina reactiva y la solubilidad de la proteína fueron reducidas (P < 0,05) al aumentar la temperatura y el tiempo de tostado. El color de las harinas fue también afectado (P < 0,05) por el tostado. En conclusión, el tostado bajo las condiciones evaluadas, puede reducir significativamente el contenido de FAN de la harina cruda de Canavalia. Sin embargo, esta respuesta positiva no incrementó el valor de EMVn de la harina cruda.

The effects of toasting on the True Metabolizable Energy, corrected to zero nitrogen retention (TMEn) and content of antinutritive factors (ANF) present in Jack Beans (JB, Canavalia ensiformis) seeds were investigated in a balance trial. Reactive lysine, protein solubility, and color were also determined in experimental meals. Raw JB meal was toasted at the following combinations of temperature and time: 180; 200; 220 and 230°C/3min; 230 and 240°C/2min; and 230 and 240°C/1min. Five caecectomised cockerels were randomly assigned to each JB meal (raw or toasted). Each bird was intubated 40g of a given meal and excreta were quantitatively collected during 72h. Concanavalin A binding to duodenal mucosa was assessed by immunohistochemistry. No hemaglutinating activity was detected in toasted JB but Con A binding to duodenal mucosa ranged from moderate to weak. Toasting JB meal at 220; 230°C/3min; and 240°C/2min reduced the original canavanine content of JB in more than 90%. TMEn value of raw JB was not improved by toasting. No significant differences were found between TMEn of raw JB and those of JB toasted at 200; 220; 230°C/3min; 240°C/1min or 240°C/2min, whereas toasting JB meal at 180°C/3min; 230°C/1min or 230°C/2min significantly (P < 0.05) reduced TMEn. Reactive lysine and protein solubility were reduced (P < 0.05) as both temperature and toasting time increased. Meal color was also affected (P < 0.05) by toasting. In summary, under the conditions tested, toasting can effectively reduce ANF content in raw JB. However, this positive response did not improve the TMEn value of raw JB.

Study on chemiluminescence of human peripheral blood lymphocytes / 中国免疫学杂志

Lymphocyte chemiluminescence (Ly-CL) is one of the early events involved in the activation of lymphocytes. This CL is thought to be result ed from the reactive oxygen species (O_2, H_2 O_2, OH, 'O_2) produced by the lymphocytes, the consequent oxidation of luminol and emission of light. In this paper we investigated the experimental condition for ConA-induced human peripheral blood lymphocyte (PBL) CL by using orthogonal design. The effects of new born calf serum (NCS), bovine serum albumin (BSA), temperature and time of cell storage on CL were also studied. The results show that the optimal condition of Ly-CL assay was 1.0 ml PBL suspension (1.4 ? 10~6 cell/ml ), 0.2 ml Luminol solution ( 7 ? 10~(-4)M) and 0.2 ml ConA ( 700 ?g /ml ). The isolated PBL were suspended in phenol red-free Hanks solution containing 0.1% BSA and can be kept for 2 hr at 4℃ before being used without adversely affecting cell viability and CL.

Fluorescence studies on concanavalin-A.

Using the lectin-concanavalin-A, the tryptophan fluorescence as a function of pH was studied. The pH dependent, fluorescence intensity changes were significantly higher when excited at 305 nm, than when irradiated at 280nm. Only one tryptophanyl per monomer of concanavalin-A was available for oxidation by N-bromosuccinimide in the dimeric form at pH 4·9; no tryptophanyl could be oxidised in the demetallised dimer (pH 3·0) and native tetramer (pH 7·0). Based on this fluorescence data and the already known crystal structure data, it appears that tryptophanyl 88 in concanavalin-A may be selectively excited by 305 nm radiation.