Construction and preservation of a stable and highly expressed recombinant Helicobacter pylori vacuolating cytotoxin A with apoptotic activity

Background H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer. Results A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA34–854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay. Conclusions A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

Construction and Preservation of a Stable and Highly Expressed Recombinant Helicobacter pylori Vacuolating Cytotoxin A with Apoptotic Activity

Background: VacA is the only exocrine toxin of H. pylori, which plays a very important role in the customization of H. pylori in the gastric mucosa, contributing to the pathogenesis of gastritis-cancer. Studies about vacA structure and function was hindered by the lack of efficient production system. In this study, we developed methodology for expression, purification and stable storage of a functionally active vacA toxin in E.coli.Results: A 2502-bp fragment and vacA gene were identified. An 89.7-kDa VacA34-854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.Conclusions: A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

Identification and Quantification of Sodium Benzoate in Different Brands of Mango Juices Available in Tangail Region, Bangladesh

Chemical preservation has become an increasingly important practice in modern food technology. Sodium benzoate is a permitted food additive in restrictive amounts by international laws, but their content must be declared and must not exceed the established limits by legislation. An experimental study for the level of sodium benzoate in different brands of mango juices available in the markets, stores and shops in Tangail region of Bangladesh was determined by high performance liquid chromatography. A Luna 5 ? C18 (2) 100A column (250 × 4.6 mm) was used for the chromatographic analysis. Chromatographic separation was achieved with isocratic solvent system comprising of sodium acetate and acetic acid buffer (pH =4.0)/acetonitrile in the ratio of 80:20 (1 ml/min) at 37oC and the chromatograms were recorded at 254 nm. The limit of detection and quantification for sodium benzoate was 0.00076 mg/100 ml and 0.00231 mg/100 ml, respectively. Quantification of the selected brand juices revealed that the level of the used sodium benzoate was within the FDA standard range. But by comparing with the Bangladesh Standard and Testing Institute (BSTI), brand-1 and brand-3 of the analyzed juice samples was found to deviate the current legal limits. The percentage recovery was found to be 92.04 ± 1.98 to 98.01 ± 1.91. It was found that some of the brands used excess amount of sodium benzoate which may be harmful for our health.Bangladesh Pharmaceutical Journal 20(1): 20-26, 2017

Dissolution Behavior of Zinc from Gel Composites Consisting of Calcium Phosphate and Alginate

Amorphous calcium phosphate (ACP) is known as one of the precursors of bone hydroxyapatite (HAp). The ACP transforms into low crystalline HAp around 40°C. Zinc (Zn) is an essential trace element for the human body and has an important role in bone formation. Osteoporosis has been linked to Zn deficiency. Biomaterials that release Zn at site of bone resorption can potentially inhibit the progression of osteoporosis. In this study, we successfully prepared Zn-releasing injectable materials that consisting of ACP and alginate gel. We investigated the Zn dissolution behavior from these composite samples in environment that imitate bone metabolism. The Zn containing gel released Zn into an acetic acid buffer, which mimics the environment of osteoclast activity. In contrast, the gel sample did not released Zn when soaked in a simulated body fluid (Kokubo’s solution), a solution that mimics the environment of osteoblast activity and cell quiescence. Therefore, the ACP/alginate gel composite that is cross-linked with Zn2+ could control the rate of release in Zn. These gels are expected to be a Zn controlled-release intelligent material.

Synthesis of Zinc-Containing Amorphous Calcium Phosphate

Zinc is an essential trace element in body and has an important role of bone formation. Osteoporosis occurs by imbalance of osteoclasts and osteoblasts activity. The osteoclasts activity lowers the pH of peripheral body environment. Therapeutic agents release material in response to the osteoclasts activity is expected to be a controlled released material. In this study, Zn-containing amorphous calcium phosphate (ACP) powder was synthesized by wet synthesis. Zn content of obtained powders was higher than that of beginning content. ACP powders could be easy to take Zn at wet synthesis. After soaking in phosphate buffered saline for 24 hours, all of synthetic powders were transformed into low crystalline apatite. On the other hand, after soaking in acetic acid buffer for 24 hours, all these powders dissolved. Zn-containing ACP powders are expected to be a Zn controlled released material.

PREPARATION OF SPHERICAL GRANULES OF OCTACALCIUM PHOSPHATE FOR MEDICAL APPLICATION

Octacalcium phosphate (OCP) is regarded as a precursor of hydroxyapatite (HA) which is an inorganic constituent of human bones and teeth. OCP is also becoming regarded as one of the important biomaterials. Despite some studies on OCP as biomedical materials, there are few methods for shape forming of OCP. The objective of this study is preparing spherical granules of OCP. The spherical granular shape has an advantage for handling. The spherical granules can achieve easy injection into the defect site by a catheter. In the present study, preparation of spherical granules of OCP from α-tricalcium phosphate (α-TCP) was attempted. The starting material of α-TCP powder was dispersed in the gelatin solution. The resultant slurry was added into vegetable oil, and then the spherical granules of α-TCP/gelatin were formed by the surface tension of the slurry and the shearing force of stirring. By calcining the obtained α-TCP/gelatin granules, the spherical granules with α-TCP single phase were obtained. These spherical granules of α-TCP were immersed in the acetic acid buffer solution whose temperature and pH were controlled. The calcium phosphate spherical granules containing OCP were obtained. The shorter treatment time was favorable for preparing spherical granules containing more OCP.

Development and Validation of a Reversed-Phase Ultra-Performance Liquid Chromatographic Method for Assay of Lacidipine and Related Substances

A simple isocratic, RP-ultra-performance LC method was developed and validated for the determination of lacidipine, three process impurities formed during synthesis, and three degradation products present in drug substance and the drug product. An efficient chromatographic separation was achieved on an Acquity BEH C18 column using pH 4.5 ammonium acetate–acetic acid buffer–methanol (70 + 30, v/v) mobile phase. The monitoring wavelength was 240 nm, and the flow rate 0.25 mL/min. Forced degradation studies using acid, alkali, peroxide, water, heat, and light were conducted, and all impurities were separated. The method was validated successfully for specificity, precision, linearity, accuracy, LOD, LOQ, and robustness, according to International Conference on Harmonization guidelines. The linearity of the calibration curve for lacidipine and each impurity was found to be very good (r2 > 0.999). This method is shown to be suitable for analysis of lacidipine to evaluate the quality of drug substance and a drug product.