[Corneal neovascularization: review article]

Corneal neovascularization occurs through inordinate wound healing after infection, injury or surgery. Neovascularization is formation of new vascular structures in the locations which had not already vessels. The two overlapping mechanisms including vasculogenesis and angiogenesis are probably involved in neovascularization process, and the last mechanism is more involved in tumor growth and corneal and retinal disorders. In fact, corneal neovascularization is a visual threatening status that usually occurs along with inflammatory or infectious disorders of the eye surface. The studies of angiogenesis-related cancer showed that there is a balance between angiogenic factors [such as VEGF and FGF] and antiangiogenic molecules [such as angiostatin, endostatin and pigment epithelium-derived factor; EPDF] in cornea. Problems such as inflammation, infection, injury and lesions result in corneal neovascularization, which are due to stimulation of angiogenesis in this tissue. Corneal neovascularization may be influenced by matrix inetalloproteinase [MMPs] and other proteolytic enzymes. The application of new medical and surgical therapies such as angiostatic steroids, non-steroidal anti inflammatory drugs, argon laser photocoagulation and photodynamic therapy [PDT] in animal models had been efficient to some extent for inhibition of corneal neovascularization. In this study we reviewed neovascularization-dependent corneal disorders and molecular processes involved in this disorder, and also their potential therapies

[Bovine casein hydrolysis using green Ficus carica extract]

The problem of casein digestion is seen in some cases specially, among infants. In this study, it is attempted to consider the hydrolysis of cow's milk casein by means of Ficin enzyme in green Fig extract. Cow's milk casein intolerance occurs in a considerable number of infants and without early diagnosis and food replacement can lead to complications like malnutrition and growth retardation. Hydrolysis of cow's milk casein by chemical and enzymatical reactions is one of the main strategies for production of hydrolyzed milk for allergic infants. In this study there has been an effort to use green fig proteases for cow's milk hydrolysis as a new and cheap source. The aqueous extract was prepared from green fruit of F. carica using phosphate buffered saline. To evaluate the degree of hydrolysis, different concentrations of extract were added to casein and incubated for 1, 3 and 6 hours at various conditions including four buffering system including tris-HCl [pH 8.5] phosphate [pH 7], citrate [pH 5.5] and acetate [pH 4.5] buffers. Hydrolysis of casein was assessed by SDS-PAGE and band densitometry estimated using Scion lab soft ware. The results indicated that fig extract could hydrolyze pure casein completely at ratio 1/100 [enzyme to substrate] for one or three hours and at 1/500 hydrolyzed casein partially at 6 hours at phosphate buffer. Therefore, phosphate buffer system is more suitable than other buffer systems for casein hydrolysis. The results of this study showed that ficin can be used as a potential protease for the hydrolysis of milk casein which is used for nutritional targets

[Determination of Helicobacter pylori immunogenes using two- dimensional gel electrophoresis and immunoblotting]

Helicobacter pylori is one of the most common infectious agents that colonizes in the mucus layer of stomach. This bacterium has been identified to be the etiologic agent of chronic active gastritis, peptic ulceration and gastric cancer. The present study was aimed to identify H. pylori immunogenes for clinical diagnisis of the infection in the above 3 groups of patients. H. pylori bacteria isolated from biopsy specimens of patients suffering from gastritis, peptic ulcer and gastric cancer were extracted in an extraction solution containing lysozyme, urea and CHAPS. Two-dimensional gel electrophoresis were performed. The resolved proteins were transferred to PVDF membrane using tank blotting and their reaction with purified IgG fraction of the patients. Sera were determined by immunoblotting. The bacterial extract showed several hundreds of silver-stained spots with molecular weights [MW] ranging from 10 to 100 KDa and isoelectric points [pI] ranging from 3.5 to 9.5. This pattern contained 6-7 major proteins, some of which as protein groups consisted of several spots. The results of immunoblots revealed that several protein spots with different MW and pI, were stained with all three groups of patients. sera but some proteins were stained only with one or two groups of sera. The protein spot with MW of 30 KDa reacted with sera of only two groups of patients; gastritis and gastric cancer; the protein with MW of 18 KDa reacted only with sera of gastritis patients. These proteins can be potential candidates for recognition of the type of gastric disorder. In addition, the results indicated that protein profiles of H. pylori, isolated from gastric cancer and peptic ulcer, are more similar to each other, comparing to that of gastritis patients

[Isolation and primary culture of rat thymic epithelial cells using actinidin enzyme of kiwi fruit]

Proteolytic enzymes specially collagenase are used for degredation of extracellular matrix, cell isolation and primary cell culture. It is important to substitute a low cost and easily obtained plant or animal protease for collagenase. In the present study the enzyme actinidin which is found abundantly in kiwi fruit, was used to isolate thymic epithelial cells from rat thymus. Actinidin with different concentrations [from 1 to 10 mg/ml] and at different times [3, 4 or 5h] was used to isolate rat thymic epithelial cells. The isolated epithelial cells were cultured on collagen coated dishes in Willam's E medium. The percentage of viable isolated cells was estimated by the trypan blue test and morphology of the cells examined microscopically after staining with Papanicoloau. Actinidin with a concentration of 4 mg/ml for 3.5-4 h digested extra-cellular matrix of rat thymus and isolated thymic epithelial cell appropriately. The rate of viability of the separated cells was estimated 90-95% in all isolates. The results of this study indicated actinidin is comparable to collagenase in isolation of epithelial cells from the thymus of rat and probably other animals. Considering its simpler purification and its low cost, this enzyme is a suitable and sale substitute for collagenase which can be used for isolation of thymic epithelial cells from rat and probably other animals

Surveying the effect of 100 kD protein of shark cartilage on erythroleukemic K[562] cell line and normal.lymphocytes

Shark cartilage is used in complementary medicine for the removal of tumor in cancer patients. However, scanty reports have addressed the effect of 100 KDa fraction on immunity system. The aim of the present study was to evaluate the effect of this fraction in human erythroleukemic K[562] cell line and normal lymphocytes. Gel chromatography purification strategy was employed for purification of 100 KDa fraction from the shark cartilage. Erythroleukemic K[562] cell line and normal lymphocytes were cultured in RPMI 1640 [Sigma], supplemented with 10% fetal calf serum, peniciline- streptomycin, L-glutamine and incubated at 37 °C for 2 days. Normal mononuclear peripheral blood cells were collected by ficole. Cellular vital capacity was determined by MTT. During MTT, erythroleukemic K[562] cell line revealed to have a meaningful suppression at 15 micro g protein concentration when compared with controls [p<0.0l]. To our knowledge, it is the first study that demonstrates l00kD protein of shark cartilage to induce cell death in erythroleukemic K[562] cell line in vitro

Purification of Kunitz soybean trypsin inhibitor using affinity chromathography

soybean trypsin inhibitor, a single chain protein with 181 amino acids and two disulfide bonds, has special tendency for pancreatic trypsin enzyme. This protein exists in soybean abundantly and showes high resistance to heating and chemicals. Various procedures such as combination of different chromatographic methods, immobilized metal affinity chromatography and preparative electrophoresis have been used to purify this protein. Pulverized soybean was defatted with methanol and the remainder was extracted after being solved in dionized water. Storage proteins in solution was eliminated by use of isollectric precipitation method. Trypsin inhibitor riched fraction was adsorbed on an affinity column with trypsin as the ligand. After washing unbonded proteins, trypsin inhibitor was eluted from the collumn by decreasing pH. Purity and activity of the inhibitor was assessed by SDS-PAGE and an spectrophotometric method, respectively. SDS-PAGE demonstrated a protein with a single band and molecular weight of 20 KDa with purity of more than 99 percent under reducing and non-reducing conditions. Activity of the purified inhibitor protein was 3600 TlU/mg. The results showed that overloading of the affinity collumn to gether with chromatography at pH of 6 to 8 increased the purity. The proposed method is simple and efficient for preparation of large amounts of pure trypsin inhibitor. In addition, this method does not need any further steps such as dialysis