Cloning and Expression of Recombinant Human Epidermal Growth Factor In Escherichia Coli

The expression of recombinant hEGF (human epidermal growth factor) in Escherichia coli was conducted to produce hEGF with free of inclusion bodies and biologically active. The recombinant hEGF was constructed using sticky ends ligation and resulted in successful insertion of the hEGF gene into the multiple cloning sites of the pFLAG-ATS. This insertion was confirmed by restriction enzyme analysis, PCR (polymerase chain reaction) and DNA (deoxyribonucleic acid) sequencing with 100% homology. The recombinant hEGF was expressed in E. coli at 2, 4 and 6 hours with induction of 0.5 mM and 1.0 mM IPTG (isopropylthiogalactopyranoside).This recombinant was found to be expressed as periplasmic fraction and whole cell insoluble fraction as confirmed with SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and western blotting analyses. These analyses showed the molecular weight of hEGF was approximately 6.8 kDa. There was no significant difference in the production of hEGF when the expression was induced with different IPTG concentration; however, for each IPTG concentration, there was significant difference between 0 hour and all the post-induction hours. In addition, hEGF was found to be significantly higher in periplasmic fraction as compared to the whole cell insoluble fraction (196.5 ng/ml as compared with 167 ng/ml at 2 hour, 175.7 ng/ml as compared with 115.3 ng/ml at 4 hour and 168.3 ng/ml as compared with 140 ng/ml at 6 hour). Growth-stimulating activity of periplasmic hEGF was studied using BrdU (bromodeoxyuridine) cell proliferation assay and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) cell proliferation assay. The periplasmic hEGF as compared to the standard hEGF was found to be biologically active and showed similar activity. There were significant findings of periplasmic hEGF in stimulating the growth of HEK (human epidermal keratinocytes) at 24, 48 and 72 hours of incubation. Even at the highest concentration of periplasmic hEGF (10 ng/ml), the growth-stimulating activity still occurred and incubation at 48 hour resulted with the highest stimulation (85.4% at 10 ng/ml, 77.7% at 1 ng/ml, 70.7% at 0.1 ng/ml, 55.9% at 0.01 ng/ml and 37.6% at 0.001 ng/ml). Besides, MTT cell proliferation assay of periplasmic hEGF on HDF (human dermal fibroblasts) showed significant increased in the growth-stimulating activity when the duration of incubation increased. Highest percentage of HDF growth was found at 72 hour incubation as compared with 24 and 48 hours incubation. In conclusion, the findings showed recombinant hEGF was successfully expressed in E. coli and the growth-stimulating activity of hEGF was determined

Cruciferous vegetables: dietary phytochemicals for cancer prevention

Relationships between diet and health have attracted attention for centuries; but links between diet and cancer have been a focus only in recent decades. The consumption of diet-containing carcinogens, including polycyclic aromatic hydrocarbons and heterocyclic amines is most closely correlated with increasing cancer risk. Epidemiological evidence strongly suggests that consumption of dietary phytochemicals found in vegetables and fruit can decrease cancer incidence. Among the various vegetables, broccoli and other cruciferous species appear most closely associated with reduced cancer risk in organs such as the colorectum, lung, prostate and breast. The protecting effects against cancer risk have been attributed, at least partly, due to their comparatively high amounts of glucosinolates, which differentiate them from other vegetables. Glucosinolates, a class of sulphur- containing glycosides, present at substantial amounts in cruciferous vegetables, and their breakdown products such as the isothiocyanates, are believed to be responsible for their health benefits. However, the underlying mechanisms responsible for the chemopreventive effect of these compounds are likely to be manifold, possibly concerning very complex interactions, and thus difficult to fully understand. Therefore, this article provides a brief overview about the mechanism of such compounds involved in modulation of carcinogen metabolising enzyme systems

Apoptosis as a mechanism of the cancer chemopreventive activity of glucosinolates: a review

Cruciferous vegetables are a rich source of glucosinolates that have established anti-carcinogenic activity. Naturally-occurring glucosinolates and their derivative isothiocyanates (ITCs), generated as a result of their enzymatic degradation catalysed by myrosinase, have been linked to low cancer incidence in epidemiological studies, and in animal models isothiocyanates suppressed chemically-induced tumorigenesis. The prospective effect of isothiocyanates as anti-carcinogenic agent has been much explored as cytotoxic against wide array of cancer cell lines and being explored for the development of new anticancer drugs. However, the mechanisms of isothiocyanates in inducing apoptosis against tumor cell lines are still largely disregarded. A number of mechanisms are believed to be involved in the glucosinolate-induced suppression of carcinogenesis, including the induction of apoptosis, biotransformation of xenobiotic metabolism, oxidative stress, alteration of caspase activity, angiogenesis, histone deacytylation and cell cycle arrest. The molecular mechanisms through which isothiocyanates stimulate apoptosis in cancer cell lines have not so far been clearly defined. This review summarizes the underlying mechanisms through which isothiocyanates modify the apoptotic pathway leading to cell death

Naturally-occurring glucosinolates, glucoraphanin and glucoerucin, are antagonists to aryl hydrocarbon receptor as their chemopreventive potency

As a cytosolic transcription factor, the aryl hydrocarbon (Ah) receptor is involved in several patho- physiological events leading to immunosuppression and cancer; hence antagonists of the Ah receptor may possess chemoprevention properties. It is known to modulate carcinogen-metabolising enzymes, for instance the CYP1 family of cytochromes P450 and quinone reductase, both important in the biotransformation of many chemical carcinogens via regulating phase I and phase II enzyme systems. Utilising chemically-activated luciferase expression (CALUX) assay it was revealed that intact glucosinolates, glucoraphanin and glucoerucin, isolated from Brassica oleracea L. var. acephala sabellica and Eruca sativa ripe seeds, respectively, are such antagonists. Both glucosinolates were poor ligands for the Ah receptor; however, they effectively antagonised activation of the receptor by the avid ligand benzo[a]pyrene. Indeed, intact glucosinolate glucoraphanin was a more potent antagonist to the receptor than glucoerucin. It can be concluded that both glucosinolates effectively act as antagonists for the Ah receptor, and this may contribute to their established chemoprevention potency

Allergens derived from shrimp

Allergy caused by food is usually type 1 allergy of four types of allergic reactions. One of the most widespread allergic is those that are caused by crustacean shellfish. Crustaceans are classified among arthropods which include crab, crayfish, lobster, prawn and shrimp. Shrimp which are broadly consumed as nutritional food is one of the most important food that contribute to allergy. Thus, reducing the allergenicity of shrimp allergen will be helpful to individuals who are sensitive to shrimp and for this reason the characteristics of each allergen need to be studied. Those sensitized individuals can develop urticaria, angiodema, laryngospasm, asthma and life threatening anaphylaxis. To date, four main allergens contribute to allergic reactions. They are tropomyosin (TM), a highly conserved and heat stable myofibrillar protein of 35-38 kDa followed by arginine kinase (AK) which is also known as Pen m 2 or Lit v 2 with 40 kDa. Two other contributing allergens are sarcoplasmic calcium-binding protein (SCP) also known as Lit v 4 with 22 kDa and myosin light chain (MLC) which is also termed as Lit v 3 with 20 kDa. This mini-review will provide a better understanding of each allergen derived from shrimp which subsequently will help to reduce the allergenicity

Polycyclic aromatic hydrocarbons (PAHs) and their bioaccessibility in meat: a tool for assessing human cancer risk

Polycyclic aromatic hydrocarbons (PAHs) are primarily formed as a result of thermal treatment of food, especially barbecuing or grilling. Contamination by PAHs is due to generation by direct pyrolysis of food nutrients and deposition from smoke produced through incomplete combustion of thermal agents. PAHs are ubiquitous compounds, well-known to be carcinogenic, which can reach the food in different ways. As an important human exposure pathway of contaminants, dietary intake of PAHs is of increasing concern for assessing cancer risk in the human body. In addition, the risks associated with consumption of barbecued meat may increase if consumers use cooking practices that enhance the concentrations of contaminants and their bioaccessibility. Since total PAHs always overestimate the actual amount that is available for absorption by the body, bioaccessibility of PAHs is to be preferred. Bioaccessibility of PAHs in food is the fraction of PAHs mobilized from food matrices during gastrointestinal digestion. An in vitro human digestion model was chosen for assessing the bioaccessibility of PAHs in food as it offers a simple, rapid, low cost alternative to human and animal studies; providing insights which may not be achievable in in vivo studies. Thus, this review aimed not only to provide an overview of general aspects of PAHs such as the formation, carcinogenicity, sources, occurrence, and factors affecting PAH concentrations, but also to enhance understanding of bioaccessibility assessment using an in vitro digestion model

A glucosinolate-rich extract of Japanese Daikon perturbs carcinogen-metabolizing enzyme systems in rat, being a potent inducer of hepatic glutathione S-transferase.

Purpose: Glucosinolates/isothiocyanates are an established class of naturally occurring chemopreventive agents, a principal mechanism of action being to limit the generation of genotoxic metabolites of chemical carcinogens, as a result of modulation of cytochrome P450 and phase II detoxification enzymes. The objective of this study was to assess whether a glucosinolate-rich extract from Daikon sprouts, containing glucroraphasatin and glucoraphenin, is a potential chemopreventive agent by modulating such enzymes in the liver and lung of rats. Methods: Rats were exposed to the glucosinolate-rich Daikon extract through the diet, at three dose levels, for 14 days, so that the low dose simulates dietary intake. Results: At the low dose only, a modest increase was noted in the hepatic dealkylations of methoxy-, ethoxy-, pentoxyresorufin and benzyloxyquinoline that was accompanied by elevated expression of CYP1 and CYP3A2 apoprotein levels. In lung, only a modest increase in the dealkylation of pentoxyresorufin was observed. At higher doses, in both tissues, these increases were abolished. At the same low dietary dose, the Daikon extract elevated markedly glutathione S-transferase activity paralleled by rises in GSTα, GSTμ and GSTπ protein expression. An increase was also noted in quinone reductase activity and expression. Finally, glucuronosyl transferase and epoxide hydrolase activities and expression were also up-regulated, but necessitated higher doses. Conclusion: Considering the ability of Daikon glucosinolates to effectively enhance detoxification enzymes, in particular glutathione S-transferase, it may be inferred that consumption of this vegetable may possess significant chemopreventive activity and warrants further evaluation through epidemiology and studies in animal models of cancer

Flat Ge-doped optical fibres for food irradiation dosimetry

Exposing food to radiation can improve hygiene quality, germination control, retard sprouting, and enhance physical attributes of the food product. To provide for food safety, radiation dosimetry in irradiated food is required. Herein, fabricated germanium doped (Ge-doped) optical fibres have been used. The fibres have been irradiated using a gamma source irradiator, doses in the range 1 kGy to 10 kGy being delivered. Using Ge-doped optical fibres of variable size, type and dopant concentration, study has been made of linearity, reproducibility, and fading. The thermoluminescence (TL) yield of the fibres were obtained and compared. The fibres exhibit a linear dose response over the investigated range of doses, with mean reproducibility to within 2.69 % to 8.77 %, exceeding the dose range of all commercial dosimeters used in evaluating high doses for the food irradiation industry. TL fading of the Ge-doped flat fibres has been found to be < 13%

Antibacterial activity of Boesenbergia rotunda (L.) Mansf. A. extract against Escherichia coli

An awareness of Escherichia coli as a foodborne pathogen and illness causing bacterium has been increased among consumers. Moreover, there is demand for natural product in order to reduce synthetic product that can cause toxic to the human. In this study, antibacterial activity, in term of MIC, MBC and killing-time curve of methanolic extract of Boesenbergia rotunda have been tested against a standard E. coli ATCC 25922 and two E. coli isolated from milk products using Clinical and Laboratory Standard Institute (CLSI) methods. The results show that B. rotunda extract was susceptible to all E. coli strains. The MIC and MBC values of B. rotunda extract against E. coli ranged 0.019 mg/mL 2.5 mg/mL and 0.039 mg/mL – 5.0 μg/mL, respectively. Killing-time curves were constructed at concentrations of 0x MIC, 1/2x MIC, 1x MIC, and 2x MIC. All E. coli strains can be killed with concentration of 2x MIC after 2 hours. The results show that B. rotunda extract has potential antibacterial activity against E. coli

Aflatoxin M1 in milk and dairy products, occurrence and recent challenges: a review

Background Milk is a highly nutritious food, and it is a source of necessary macro- and micronutrients for the growth, development and maintenance of human health. However, it may also be a source of natural food contaminants that may cause disease. The presence of aflatoxin M1 (AFM1) in milk and dairy products throughout the world has been known since twenty to thirty ago. Milk and dairy products contamination with aflatoxin M1 is important problem worldwide especially for developing countries for the last ten to twenty years. Scope and Approach The presence of this mycotoxin in these products is important issue, especially for children and infants, who are more susceptible than adults. This review provides information regarding the occurrence of AFM1 in milk and dairy products in many regions of the world, its stability during processing and some reduction strategies. In this review the toxicity, occurrence of AFM1 in milk and dairy products (preferably for the last 5 years), regulations, strategies for its reduction, latest developments in detection methodologies and future challenges are described. Key Findings and Conclusions Strict regulations and adapting good storage practices in developed countries have minimized the contamination of AFM1 in milk and dairy products. The current advancements in analytical techniques have helped the law enforcement agencies to implement strict regulations. Furthermore, the improvement in analytical facility and increasing the awareness related to the health effects of AFM1 in milk and dairy products could minimize its occurrence level in developing countries