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  • br Materials and methods br Results and

    2020-01-16


    Materials and methods
    Results and discussion
    Conclusions Clitocypin is another member of the mycocypin family of cysteine protease inhibitors that is unique to Prochlorperazine australia shown to exhibit strong insecticidal activity against Colorado potato beetle larvae. This effect is concentration dependent and is greater against younger larvae. High Prochlorperazine australia Clt concentration in the diet increased larval mortality and reduced weight gain while lower concentrations reduced weight gain without affecting larval survival. Furthermore, these effects on CPB larvae were observed for clitocypin isolated from fruiting bodies, its recombinant variants expressed in E. coli and in transgenic potato. The negative effect was observed, even at very low expression levels, in potato leaves (~0.03% of total soluble protein), when larvae were exposed from hatching onwards. The mode of action is shown to be antinutritional by its strong inhibition of digestive cysteine proteases in larval guts. Optimization of transgene expression levels in plants would greatly enhance the insecticidal effectiveness of clitocypin used as a biopesticide. As a cysteine protease inhibitor it could be evaluated as a control agent for other pests utilizing cysteine proteases for digestion and it could also contribute to plant resistance to other stress conditions, biotic and abiotic, in which proteases play important roles [22]. Confirmation of the strong antinutritional potential of mycocypins by another member of the superfamily establishes mushrooms as an attractive source for novel bioactive proteins.
    Introduction Chikungunya virus (CHIKV) is a reemerging arthropod-borne virus, which belongs to the Alphavirus genus of the Togaviridae family. CHIKV was first isolated from the Makonde Plateau of the southern province of Tanzania in 1952 [1], where it was believed to be maintained in a sylvatic transmission cycle among the mosquitoes and non-human primates of the region [2]. CHIKV, which is spread majorly by Aedes sp. of mosquitoes, Aedes aegypti and Aedes albopictus, is an etiological agent of Chikungunya fever in humans. It causes an abrupt onset of fever upon infection, which is characterized by maculopapular rash, incapacitating arthralgia, myalgia, headache and chill [3]. Chikungunya fever is also characterized by high viraemic content with the concomitant irregularities such as intensified lymphopenia and thrombocytopenia [1]. Despite its first emergence in the early 1950s, CHIKV reemerged in the mid of first decade of twenty-first century in several islands of the Indian Ocean where epidemic of an unprecedented magnitude broke out. These outbreaks were found to be associated with an unexpected mutation (A226V) in the E1 structural protein of the virus, which enhanced the viral infectivity in one of its major carrier mosquito A. albopictus [2]. Indian Ocean CHIKV outbreak was succeeded by many outbreaks in various geographic locations in Indian subcontinent and Caribbean Sea Islands [4]. A large number of patients were infected from CHIKV in Southern regions of India and a minor CHIKV outbreak in several parts of North-East India was reported in 2011 [5,6]. In 2013 again, large numbers of chikungunya infections were reported in the various Caribbean islands and French Guiana [4]. CHIKV belongs to the genus alphavirus, which includes other pathogenic viruses like Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), the western, eastern and Venezuelan equine encephalitis virus (VEEV) etc. These are enveloped, positive-sense ssRNA viruses that are responsible for causing diseases in humans and animals. The cryo-electron microscopy (cryo-EM) structures of alphaviruses have revealed that the virus particles are spherical in shape of 65–70 nm in diameter having icosahedral symmetry and a triangulation number four (T = 4) [7]. The genome comprises of an approximately 11.8 kb (+)-sense ssRNA with a 5′ cap and a 3′ poly(A) tail [8]. Upon viral entry into the host cell, the 5′ two-third of the viral genome RNA is directly translated into the nonstructural polyprotein precursor nsP1234, which is further processed by the proteolytic catalysis of virus encoded cysteine protease (nsP2) into separate nonstructural proteins (nsPs) viz. nsP1, nsP2, nsP3 and nsP4. These proteins with the help of host factors contribute to architect the replication complexes in order to replicate the viral genome and transcribe the 26S subgenomic viral RNA. The subgenomic RNA codes for the structural polyprotein containing capsid (CP), E3, E2, 6K & E1 [9,10].