A novel assay method for calcium calmodulin dependent phosphatase from bovine brain extract.

Calcium calmodulin dependent protein ser/thr phosphatase, also referred to as protein phosphatase 2B (PP2B), is rich in neural tissue, and plays an important role in the overall function of the nervous system. Routinely phosphatase assay employs, para-Nitrophenlylphosphate (p-NPP), as a substrate, is also extended to assay PP2B. However, in the present study, the differential spectral characterstic property of tyrosine and phopshotyrosine has been exploited to employ the latter as a candidate substrate for the PP2B assay. The specific activity of PP2B using phosphortyrosine in bovine Bos Taurus indicus brain extract (Bos Taurus indicus), was measured in presence of different metal ions like Ca2+, Mn2+ and Mg2+. Further modulators like dithiothreitol (DTT), calmodulin (CaM) and metal chelators such as EGTA and EDTA were applied to confirm the role of divalent cations and to determine calcium calmodulin dependent phoshphatase activity. PP2B activity was higher with phosphotyrosine in presence of Ca2+ than with p-NPP. Further experiments, involving calmodulin as a modulator, confirmed phosphotyrosine as a better substrate over p-NPP. Calmodulin further enhanced the effect of phosphotyrosine as a potential substrate confirming calcium calmodulin dependent phosphatase activity. Phosphotyrosine is proposed as a better substrate in assaying calcium dependent phosphatase activity when compared to para-nitrophenylphosphate.

Physiological and biochemical characterization of egg extract of black widow spiders to uncover molecular basis of egg toxicity

BACKGROUND: Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity. RESULTS: In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na+, K+and Ca2+ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed. CONCLUSIONS: The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs' toxic actions.

Amino and fatty acid compositions in Haruan traditional extract (HTE) / Composición de aminoácidos y ácidos grasos en extractos de Haruan tradicional

Evaluation of amino and fatty acids compositions in Haruan Traditional Extracts (HTE) was done using HPLC and GC methods. The HTE contained at least 17 amino acids with glutamic acid, glycine, leusine, aspartic acid, proline, alanine and arginine are the most, with values 1.87 - 43.13 mg/g, 21.80 - 80.85 mg/g, 7.85- 40.19 mg/g, 13.85 - 44.07 mg/g, 9.49 - 45.46 mg/g, 11.38 - 35.25 mg/g and 5.99 - 21.79 mg/g, respectively. Meanwhile, the highest percentage of fatty acids is palmitic acid; 3.54 - 26.84 percent of total protein. The others major fatty acids are stearic acid, oleic acid and linoleic acid with values 3.25 - 15.90 percent, 1.40 - 27.68 percent, 0.51 - 7.82 percent of total protein, respectively. HTE also found to have 4 extra bioactive compounds labelled as 1 to 4 on chromatographic tracing which in line with previously finding. It is concluded that the HTE is containing all the important amino acids plus some fatty acids, which is the basis to conduct antioxidant composition in both fresh Haruan and the HTE which was claimed to have wound healing properties. Comparative study was also carried out in various other extraction protocols, including commercial product.

Evaluación de las composiciones de aminoácidos y ácidos grasos en Haruan Extractos tradicional (HTE) se realizó mediante métodos de HPLC y GC. La HTE contenía al menos 17 aminoácidos con ácido glutámico, glicina, leucina, ácido aspártico, prolina, alanina y arginina como mayoritarios, con valores de 1.87 - 43.13 mg/g, 21.80 - 80.85 mg/g, 7.85 - 40.19 mg/g, 13.85 - 44.07 mg/g, 9.49 - 45.46 mg/g, 11.38 - 35.25 mg/g and 5.99 - 21.79 mg/g, respectivamente. Mientras tanto, el mayor porcentaje de ácidos grasos es el ácido palmítico; 3.54 - 26.84 por ciento de la proteína total. Otros ácidos grasos importantes son el ácido esteárico, ácido oleico y ácido linoleico con valores de 3.25 - 15.90 por ciento, 1.40 - 27.68 %, 0.51 - 7.82 por ciento de la proteína total, respectivamente. HTE también encontró cuatro compuestos bioactivos adicionales etiquetados de 1 a 4 en el seguimiento cromatográfico que está de acuerdo con resultados previos. Se concluye que la HTE contiene todos los aminoácidos importantes además de algunos ácidos grasos, que es la base para llevar a cabo la composición antioxidante, tanto en fresco Haruan y la HTE que se afirma poseen propiedades curativas. Estudios comparativos se llevaron a cabo con otros protocolos de extracción, incluido el producto comercial.

Role of pepsin in modifying the allergenicity of bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) fish.

The effect of pepsin digestion on the allergenicity of raw and thermally processed (boiled and fried) fish muscle extracts of two widely consumed fishes bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) was studied. Sere were collected from 110 patients who were hypersensitive to fish, as evidenced by their clinical history, symptoms and positive skin-prick test results. The various extracts after digestion with pepsin at different times of incubation were tested for specific IgE-binding activity by ELISA and immunoblotting with patients' sera. All the extracts of both the fishes retained their allergenicity as evidenced by ELISA and immunoblotting. In bhetki, maximum allergenicity was found in the pepsin-digested fried extract, whereas similar treatment decreased the allergenicity in fried mackerel. Results showed that raw as well as thermally processed allergens of both the fishes maintained strong allergenicity, even after digestion with pepsin for different time periods. The study revealed that the fish proteins played an important role in manifestation of allergy, due to their stable structure, which was retained even after pepsin and heat treatment.

Ovarian steroid sulphate functions as priming pheromone in male Barilius bendelisis (Ham.).

The study reveals that pre-ovulatory females of the fish Barilius bendelisis (Ham.) release sex steroids and their conjugates into the water and that a steroid sulphate of these compounds functions as a potent sex pheromone which stimulates milt production in conspecific males prior to spawning. Since males exposed to the purified subfraction III of the steroid sulphate fraction have increased milt volume and more spermatozoa with greater motility, the function of this priming pheromone appears to be to enhance male spawning success. High turbulence and faster water currents render the hillstream ecosystem extremely challenging for chemical communication. Therefore, ovulatory female fish secrete highly water soluble steroid sulphates for rapid pheromonal action in males. Inhibited milt volume in olfactory tract lesioned (OTL) males exposed to the steroid sulphate fraction and 17alpha,20 beta-dihydroxy-4-pregnen-3-one supports the concept that the pheromonally induced priming effect in male fish is mediated through olfactory pathways.

Identification and purification of IgE-reactive proteins in German cockroach extract

Cockroaches have been implicated as a cause of respiratory allergy in urban areas worldwide. IgE-reactive German cockroach proteins were identified with molecular weights (MWs) of 90, 66, 50, 43 and 36 KD by immunoblot analysis in both immune BALB/c mice and sensitized humans. Prominent IgE-reactive proteins were purified using FPLC by ion-exchange chromatography, gel filtration and hydrophobic chromatography. The N-terminal amino acid sequence of a purified protein with a MW of 66 KD on SDS-PAGE was Val-Thr-Leu-Lys-Lys(Val)-Met-Ile-Lys-Thr-Phe-Tyr. No homologous protein was found through a search of GenBank that indicated a novel IgE-reactive protein in German cockroach extract. Another purified protein with a MW of 36 KD reacted strongly with a monoclonal antibody against Bla g 2.