Heterologous expression, purification, and properties of a potato protein inhibitor of serine proteinases

The gene PKPI-B10 [AF536175] encoding in potato (Solanum tuberosum L., cv. Istrinskii) a Kunitz-type protein inhibitor of proteinases (PKPI) has been cloned into the pET23a vector and then expressed in Escherichia coli. The recombinant protein PKPI-B10 obtained as inclusion bodies was denatured, separated from admixtures by ion-exchange fast protein liquid chromatography (FPLC) on MonoQ under denaturing conditions, and renatured. The native protein was additionally purified by ion-exchange FPLC on DEAE-Toyopearl. The PKPI-B10 protein effectively inhibits the activity of trypsin, significantly weaker suppresses the activity of chymotrypsin, and has no effect on other serine proteinases: human leukocyte elastase, subtilisin Carlsberg, and proteinase K, and also the plant cysteine proteinase papain.     

Substrate phase state and time factor in variability of human fecal microbiota

Effects of substrate phase state and time factor on variability of human fecal microbiota were studied. It was shown that microecological system of native feces was characterized by marked time-dependent variability. It is unstable and begins to destruct after 24 hours of cultivation. The most sensitive elements of the system were bifidobacteria and Escherichia coli. Change of phase state of biotope eliminated the effect of factor limiting the microecosystem development, which allowed species of obligate and transitory microflora to freely colonize the growth substrate and interact with each other. The mentioned facts demonstrate that fecal microbiota exists in the environment of excess of growth substrate, which colonization is limited by cluster structure of biotope of native feces. It was concluded that phase state of growth substrate and duration of cultivation are important factors determining the population variability of fecal microbiota.     

An Artificial Neural Network Model to Predict the Phenology of Early-Maturing Soybean Varieties from Climatic Factors

Biophysics - Abstract—Soybean phenology is strongly influenced by temperature and day length, and phenological records clearly reflect the changes in climatic conditions. A model including...     

Two-component covalent inhibitor

Inhibitors that covalently damage proteins or nucleic acids offer great potency, but are difficult to rationally design and suffer from poor specificity. Here we outline a general concept for constructing covalent inhibitors, called the two-component covalent inhibitor (TCCI). The approach takes advantage of two ligand analogs equipped with pre-reactive groups. Binding of the analogs to the adjacent sites of a target biopolymer brings the pre-reactive groups in close proximity and causes their interaction followed by covalent damage of the target. In the present study we used light-activated pre-reactive groups to inactivate a DNA polymerase. It was found that the efficiency of a traditional single-component inhibitor was greatly reduced in the presence of a non-target protein, while the TCCI was not significantly affected. Our findings suggest that TCCI approach has advantages in inactivation of biopolymers in complex multi-component systems.     

Modulation of plant resistance to diseases by water-soluble chitosan

Low-molecular-weight water-soluble chitosan with a molecular weight of 5 kDa obtained after enzymatic hydrolysis of native crab chitosan was shown to display an elicitor activity by inducing the local and systemic resistance of Solanumi tuberosum potato and Lycopesicon esculentum tomato to Phytophthora infestans and nematodes, respectively. Chitosan induced the accumulation of phytoalexins in tissues of host plants, decreased the total content and changed the composition of free sterols producing adverse effects on infesters, activated chitinases, beta-glucanases, and lipoxygenases, and stimulated the generation of reactive oxygen species. The activation of protective mechanisms in plant tissues inhibited the growth of taxonomically different pathogens (parasitic fungus Phytophthora infestans and root knot nematode Meloidogyne incognita).     

Induction of Resistance to Phytophthora in Tubers of Transgenic Potato

Resistance of transgenic cultivars based on the expression of one or more resistance genes is sooner or later broken by pathogens whose race-producing rates are high. Thus, combining transgenesis with elicitor-induced resistance is a promising approach. The elicitor-induced resistance is based on the expression of multiple resistance genes, which can prevent the adaptation of pathogens to transgenic cultivars, maintain the stability of cultivars, and increase their lifespan. In this work, we used transgenic potato cultivars Temp and Superior transformed with Bacillus thuringiensis -endotoxin gene and Luk'yanovskii transformed with leukocyte interferon gene. Arachidonic acid (10^–8 M) and soluble chitosan (5 kDa, 100 g/ml) were used as elicitors for tuber treatment. Our data showed that pretreatment with elicitors causes a 15–25% increase in both the systemic prolonged resistance of potato tubers to Phytophthora infestansand their ability to repair mechanical damage.     

Epimerization of hydroxyl group in the lupan series of triterpenes

Two methods of obtaining of 3 alpha-betulinic acid and related compounds from their 3 beta-epimers were studied: the reaction of bimolecular substitution and the stereoselective reduction of 3-ketoderivatives. The substitution of acyloxy by formyloxy group in 3-O-tosyllupeol or of the betulin hydroxyl by benzoyloxy group resulted only in delta 2, 3-elimination products, with none of the expected products of bimolecular substitution being found. The catalytic hydrogenation of betulonic acid over Raney nickel resulted only in reduction of the isopropenyl double bond, whereas the use of 5% Ru/C gave a 60:40 mixture of epimers of dihydrobetulinic acid. Practically the same mixture of betulinic acid epimers was obtained when reducing betulonic acid with L-Selectride. The cytotoxic activity of 3 alpha-betulinic acid increased toward melanoma Bro cells and decreased toward melanoma MS cells.     

Role of proteinase inhibitors in potato protection

Mechanical damage or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that the lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.     

Epimerization of Hydroxyl Group in Lupan Series Triterpenoids

Two methods of obtaining 3-betulinic acid and related compounds from their 3-epimers were studied: the reaction of bimolecular substitution and the stereoselective reduction of 3-ketoderivatives. The substitution of acyloxy by formyloxy group in 3--tosyllupeol or of the belulin hydroxyl by benzoyloxy group resulted only in ^{2, 3}-elimination products, with none of the expected products of bimolecular substitution being found. The catalytic hydrogenation of betulonic acid over Raney nickel resulted only in reduction of the isopropenyl double bond, whereas the use of 5% Ru/C gave a 60 : 40 mixture of epimers of dihydrobetulinic acid. Practically the same mixture of betulinic acid epimers was obtained when reducing betulonic acid with L-Selectride. The cytotoxic activity of 3-betulinic acid increased toward the Bro melanoma cells and decreased toward the MS melanoma cells.