Firdose Ahmad Malik

Cloning and expression pattern of 3-dehydroecdysone 3β-reductase (3DE 3β-reductase) from the silkworm, Bombyx mori L.

Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, reproduction, etc. Ecdysone was inactivated to 3-dehydroecdysone (3DE) under ecdysone oxidase (EO), and followed by NAD(P)H-dependent irreversible reduction to 3-epiecdysteroid under 3DE 3a-reductase. On the other hand, 3-dehydroecdysone undergoes reversible reduction to ecdysone by 3DE 3β-reductase in the hemolymph. In this article, we cloned and characterized 3-dehydroecdysone 3β-reductase (3DE 3β-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L. The B. mori 3DE 3β-reductase cDNA contains an ORF 972 bp and the deduced protein sequence containing 323 amino acid residues. Analysis showed that the deduced 3DE 3β-reductase belongs to the aldo-keto reductase (AKR) superfamily, which has the NAD(P)-binding domain, indicating that the function of 3DE 3β-reductase depends on the existence of NAD(P)H. Using Escherichia coli, a high level expression of a fusion polypeptide band of approx. 40 kDa was observed. The high transcription of 3DE 3β-reductase was mainly observed in the genitalia and fatty bodies in the third day of the fifth-instar larvae, followed next in the head, epidermis, and hemocytes. The expression of 3DE 3β-reductase in the early of every instar was lower than that in the late of instar. When the titer of 3DE is low, higher expression of 3DE 3β-reductase is necessary to maintain the ecdysone titer in body through converting 3DE to ecdysone, while the 3DE titer is high, the expression of 3DE 3β-reductase showed feedback inhibition.

Cloning and characterization of the Bombyx mori ecdysone oxidase

The physiological titer of molting hormones in insects depends on relative activities of synthesis and degradation pathways. Ecdysone oxidase (EO) is a key enzyme in the inactivation of ecdysteroid. However, there are only a few reports on ecdysteroid inactivation and its enzymes in silkworm. In this study, we cloned and characterized the Bombyx mori EO (BmEO). The BmEO cDNA contains an ORF of 1,695 bp and the deduced protein sequence contains 564 amino acid residues. The deduced protein sequence contains two functional domains of glucose-methanol-choline oxidoreductase in N-terminal and C-terminal. Comparing the expression levels of BmEO in different tissues, high transcription was mainly present in hemocytes. Reduced expression of this enzyme is expected to lead to pathological accumulation of ecdysone in the hemolymph of silkworm larvae or pupae. Our data show that RNA inference of BmEO transcripts resulted in the accumulation of ecdysteroid and death of larvae or pupae. We infer that EO is a crucial element in the physiology of insect development.

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L.

The peritrophic membrane (PM) is a semi-permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin-binding proteins (BmPM-P43 and BmPM-P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C-terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM-P43 and BmPM-P41. The chitin-binding activity analysis showed that the BmPM-P43 and BmPM-P41 could bind to chitin strongly. It is concluded that BmPM-P43 and BmPM-P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin-binding protein family.

Immobilization of foreign protein in BmNPV polyhedra by fusion expression with partial polyhedrin fragments

Bombyx mori nucleopolyhedrovirus (BmNPV) produces large, proteinaceous crystal matrix named polyhedra, which occlude progeny virions which are produced during infection and protect virions from hostile environmental conditions. In this study, five overlapping N-terminal fragments of the BmNPV polyhedrin ORF were cloned and ligated with the foreign gene egfp, and five recombinant baculoviruses were constructed by BmNPV(Polh(+)) Bac-to-Bac baculovirus expression system was used to co-express the polyhedrin and fused protein. The results showed that the fusion proteins were highly expressed, and the foreign proteins fused with the 100aa fragment of polyhedrin could be embedded into polyhedra at a higher ratio. This study provides a new method for efficient preservation of useful proteins for the development of new biopesticide with toxin protein and delivery vector system of vaccines.

Demonstration of protein absorption in the intestinal epithelium of fish and mice by laser scanning confocal microscopy

Abstract Selective permeability for small proteins and oligopeptides occurs in the intestinal epithelium of many animal species and humans. Whole proteins are sometimes endocytosed and undergo partial hydrolysis in intestinal epithelial cells with the probable release of essential oligopeptides into the bloodstream. Increased permeability to certain proteins can cause asthma and other metabolic disorders. Permeable proteins have also been successfully used to deliver vaccines or drugs via oral consumption. Protein absorption has been inferred in many cases and demonstrated in some cases by histochemical, tracer, and analytical techniques. However, the nature and importance of protein absorption remains largely unknown. Here, we demonstrate the movement of two lumenal proteins (GFP: 26 kDa and OFP: 23 kDa) across the intestinal epithelium of fish and mice using laser scanning confocal microscopy. The results provide evidence that small proteins can be taken up intact by intestinal epithelial cells, even though large proteins are digested to single amino acids or protein fragments before they are absorbed. Our results suggest that it is possible to orally administer some small proteinous medicines for therapeutic purposes.