Pupa Protein Research Articles

Isolation of a novel peptide from silkworm pupae protein components and interaction characteristics to angiotensin I-converting enzyme

The hydrolysate of silkworm protein produced by acid protease (Asperqiius usamii NO. 537) contains angiotensin I-converting enzyme inhibitory activity. Four kinds of protein components were identified in silkworm pupae protein, albumin, globulin, prolamin and glutelin, and the albumin was in the highest quantity. The four component proteins were then further hydrolyzed by acid protease under the same conditions, and the hydrolysate of albumin was determined with the highest ACE inhibiting effect, followed by globulin. Albumin could be easily hydrolyzed under the aqueous conditions, and the overall albumin protein content was higher than the other three protein components. The peptide sequence “APPPKK” was determined by LC–MS/MS separation and X!Tandem software identification. The peptide inhibitory activity was 0.047 mg/mL in IC50. The peptide was bonded to Asp415, Asp453, Thr282, His 353, Glu162 in hydrogen bond to ACE active pocket by flexible docking calculations. This study indicates that silkworm pupae protein may be a suitable candidate to explore functional foods with anti-hypertension bioactivity.

Silkworm Pupae (Bombyx mori) Are New Sources of High Quality Protein and Lipid

This study was performed to evaluate the nutritional value of silkworm pupae (Bombyx mori) and the content of α-glucosidase inhibitor. The percentages of total protein and lipid contents by dry weight were 55.6 and 32.2%, respectively. Silkworm pupae protein had high levels of essential amino acids such as valine, methionine and phenylalanine. The contents of essential amino acids in silkworm pupae protein satisfied the FAO/WHO/UNU suggested requirements (2007). In addition, they also possessed n-3 fatty acids, especially α-linolenic acid (36.3%), as a major component. The 50% ethanol extract of silkworm pupae contained 1-deoxynojirimycin (DNJ), which is a potent α-glucosidase inhibitor. These results suggest that silkworm pupae are a new source of high quality protein, lipid, and α-glucosidase inhibitor.

Safety and Immunogenicity of H5N1 Influenza Vaccine Based on Baculovirus Surface Display System of Bombyx mori

Avian influenza virus (H5N1) has caused serious infections in human beings. This virus has the potential to emerge as a pandemic threat in humans. Effective vaccines against H5N1 virus are needed. A recombinant Bombyx mori baculovirus, Bmg64HA, was constructed for the expression of HA protein of H5N1 influenza virus displaying on the viral envelope surface. The HA protein accounted for approximately 3% of the total viral proteins in silkworm pupae infected with the recombinant virus. Using a series of separation and purification methods, pure Bmgp64HA virus was isolated from these silkworm pupae bioreactors. Aluminum hydroxide adjuvant was used for an H5N1 influenza vaccine. Immunization with this vaccine at doses of 2 mg/kg and 0.67 mg/kg was carried out to induce the production of neutralizing antibodies, which protected monkeys against influenza virus infection. At these doses, the vaccine induced 1:40 antibody titers in 50% and 67% of the monkeys, respectively. The results of safety evaluation indicated that the vaccine did not cause any toxicity at the dosage as large as 3.2 mg/kg in cynomolgus monkeys and 1.6 mg/kg in mice. The results of dose safety evaluation of vaccine indicated that the safe dose of the vaccine were higher than 0.375 mg/kg in rats and 3.2 mg/kg in cynomolgus monkeys. Our work showed the vaccine may be a candidate for a highly effective, cheap, and safe influenza vaccine for use in humans.

Hydrolyzates of Silkworm Pupae (Bombyx Mori) Protein is a New Source of Angiotensin I-Converting Enzyme Inhibitory Peptides(ACEIP)

Silkworm pupae protein is a good source of high quality protein. The hydrolyzates of silkworm pupae protein catalyzed by neutrase, pepsin, acidic protease (Asperqiius usamii NO. 537), flavourzyme, alcalase, and trypsin with inhibitory activity on angiotensin I-converting enzyme (ACE) were identified by HPLC. The hydrolyzates catalyzed by acidic protease exerted the highest inhibitory activity on ACE. The hydrolyzing conditions were optimized by one-factor, factional factorial (FFD), and center composite (CCD) design methods, and response surface methodology (RSM). Statistical analyses showed that regression of the second-order model equation is suitable to describe ACE inhibitory bioactivity. The predicted inhibitory activity of hydrolyzates on ACE was 73.5 % at a concentration of 2.0 mg/ml. Optimized RSM technique decreased IC(50) of hydrolyzates inhibiting ACE to 1.4 mg/mL from 2.5 mg/ml. The molecular weight of the components of the hydrolyzates with inhibitory activity on ACE varied from less than 500 to about 1000 Da by ultra-filter analysis. These studies suggest that hydrolyzates of silkworm protein contain ACE inhibitory activity that could form a potential source of ACE inhibitor drugs.

Pharmacophore-based structure optimization of angiotensin converting enzyme inhibitory peptide

Chemical feature based pharmacophore models were generated for an angiotensin converting enzyme (ACE) inhibitory peptide using the Discovery Studio 2.0 pharmacophore modeling approach. The pharmacophore hypothesis selected has five features (one negative ionizable region, one hydrogen bond donor, one hydrogen bond acceptor and two hydrophobic functional groups). Additionally, ACE inhibitory hexapeptide previously obtained from silkworm pupae protein was optimized to target the ACE based on the selected pharmacophore. The results suggest that tri-peptide (thr-val-phe) may be structural determinant of ACE activity. Docking studies further provided confidence for the validity of the selected pharmacophore model to perform structure optimization of the ACE inhibitory peptide.

Proximate, amino acid and mineral composition of pupae of the silkworm Antheraea pernyi in China

The chemical composition and the nutritional quality of protein of pupae of the silkworm Antheraea pernyi were investigated. Investigations showed that the pupal powder contained 7.6% moisture, 71.9% crude protein, 20.1% fat and 4.0% ash on a dry matter basis. The mineral analysis indicated high K content with a low Na/K ratio and low heavy metal content. The pupal protein contained 18 known amino acids, including all of the essential amino acids and sulphur-containing amino acids. Compared with the amino acid profile recommended by FAO/WHO, the pupal protein was of high quality due to its high content of essential amino acids. The results of the present study provide some technical information and suggestions for the food industry for more effective utilization of pupae of the silkworm A. pernyi.

Safety evaluation of protein of silkworm (Antheraea pernyi) pupae

The protein of silkworm pupae (PSP) has been thought to be a new available source of high quality protein that contains all the amino acids needed by the human body. The safety of PSP was evaluated systematically by a series of acute and sub-acute toxicological tests: (i) Acute toxicity test: The oral maximum tolerated dose of PSP was more than 15.0 g/kg body weight in mice, due to the absence of toxicity according to the criteria of acute toxic classifications; (ii) Mutagenicity test: PSP had no mutagenicity, as judged by a negative Ames test, mouse bone marrow cell micronucleus test and mouse sperm abnormality test; (iii) 30 days feeding study: No deaths or abnormal hematological, clinical chemical and histopathological changes and clinical signs had been found in rats when administrated PSP at 0.30, 0.75 and 1.50 g/kg/day to the rats for 30 days in each group during the test, respectively. No statistically significant differences had been found in body weights, food consumption and food efficiency of rats in each test group (P>0.05). These results indicate that PSP can be generally regarded as safe at a maximum dose of 1.50 g/kg/day in rats.

Experimental Production and Chemical Composition of Culex Mosquito Larvae and Pupae Grown in Agro-industrial Effluent

An experiment was conducted to grow larvae and pupae of Culex mosquito in palm oil mill effluent (POME), latex concentrate rubber effluent and microalgae Selenastrum gracile (isolate no. 166) as cultured in 70 l tanks. No larval growth was observed in rubber effluent and very few larvae were found in microalgal tanks. Sampled Culex larvae and pupae grew in POME tanks, and were considered for biochemical analyses. The crude protein, real protein, lipid and ash content of Culex pupae were significantly higher (p < 0.05) than the larvae of the third and the fourth instars. All the essential amino acids of pupae except threonine, valine, isoleucine and lysine were significantly higher (p < 0.05) than those of the fourth instar followed by the third instar. The polyunsaturated fatty acids (PUFAs) of pupae such as linolenic acid (18: 3n-3), ¡-linolenic acid (18: 3n-6), arachidonic acid (20: 4n-6), eicosapentaenoic acid (22: 5n-3) and docosahexaenoic acid (22: 6n-3) were significantly higher (p < 0.05) than those of the third and the fourth instars. The pupae bioaccumulated significantly higher (p < 0.05) amount of essential minerals than larvae of the fourth instar followed by the third instar. The production of Culex pupae per 20 l of POME was 670 g, which is equivalent to 33.50 kg·ton-1 . Due to presence of high micronutrients the larvae and pupae may be used as food for the rearing of post-larvae of 6- 7 days old and fry of catfish, and shrimp and prawn. The presence of chitin as body covering of larvae and pupae helps to develop the chitinuous body covering of shrimp and prawn.

Use of silkworm pupae meal as protein supplement in the nutrition of broiler chickens

Silkworm pupae meal is a silk industry by-product that in some countries is available as a local product. It contain up to 30% crude fat and 50% crude protein. The chemical score of silkworm pupae protein is 60 and tryptophan is its limiting amino acid. The silk worm pupae meal fat contains 25.7% linoleic acid (Udayasekhara Rao, 1994). Reddy et al (1991) reported that use of diets contain 5% 8% SWP so that replaced 50% of fish meal, lowered weight gain. The aim of this study was to measure the effects of substituting different levels of silkworm pupae (SWP) for soybean meal (SBM) in Arian (an Iranian strain) broiler rations.

Abstract

Silkworm pupae are edible insects with high-quality nutrition in many Asian countries, but consumption of silkworm pupae can cause severe IgE-mediated allergic disease. The aim of this study was to investigate the effect of heat, enzymatic hydrolysis and acid-alkali treatment on the allergenicity of silkworm pupa protein extract (SPPE). Heating reduced the allergenicity of SPPE when the temperature was higher than 60 °C. Spectroscopy studies suggested an unfolded conformation of SPPE with heating, dependent on temperature and time. Enzymatic hydrolysis revealed that SPPE at 25 to 33 kDa contained pepsin- and trypsin-resistant allergens. The results of acid-alkali treatment suggested that low pH can promote hydrolysis of SPPE and decrease its allergenicity. Thus, heat, enzymatic hydrolysis and acid-alkali treatment can significantly decrease the allergenicity of SPPE, with heat-, enzyme- and acid-alkali–resistant allergens at 25 to 33 kDa SPPE. This study can help in the development of methods to prepare silkworm pupa protein.

Differential Accumulation and Tissue Distribution of Mosquito Hexamerins During Metamorphosis

The pupal hexamerins were characterized for two mosquitoes representative of the culicine and anopheline families, Aedes aegypti and Anopheles gambiae. Like higher Diptera, both mosquito species express two types of hexamerins, Hex-1 and Hex-2, whose subunits are distinguished by different levels of methionine and aromatic amino acids. In A. aegypti there are two heterohexamers, AaHex-1 and AaHex-2. In A. gambiae there are two homohexamers, AgHex-1.1 and AgHex-1.2, and one heterohexamer, AgHex-2. These hexamerins are rich in aromatic residues, with 18–23% Phe+Tyr for Hex-1 subunits and 13–17% Phe+Tyr for Hex-2 subunits. In addition, both mosquito species synthesize methionine-rich Hex-1 subunits: Aedes AaHex-1 γ (8% met) and Anopheles AgHex-1.1 (3.9% met). Aedes Hex-1 and Hex-2 proteins exhibit different, stage-specific tissue distributions: AaHex-2 is the primary hexamerin of late larval hemolymph whereas AaHex-1 is the most important non-hemolymph protein of early pupae. Although both proteins are stored in the pupal fat body, peak AaHex-1 levels are 2-fold higher. Both pupal protein levels decline rapidly between 25 and 36 h after pupation. Furthermore, AaHex-1 not only reaches peak values in female Aedes pupae later than in males, but the methionine-rich AaHex-1 γ subunit level is specifically higher in females. These observations suggest different roles for Hex-1 and Hex-2 during mosquito development.

Induction kinetics of immune antibacterial proteins in pupae of Galleria mellonella and Pieris brassicae

1. 1. Pupae of Galleria mellonella and Pieris brassicae given an injection with live, non-pathogenic Enterobacter cloacae or abiotic foreign molecules induce an acquired immunity that corresponds with the synthesis of haemolymph proteins of antibacterial activity. 2. 2. This humoral defensive response which persists for several days, differs quantitatively between insect species and between the inducers used, although very different foreign bodies induced the same immune proteins in both lepidopteran insects. 3. 3. A stronger and longer lasting response was consistently noticed in pupae immunized with non-pathogenic bacterium than after sterile nutrient broth injections. 4. 4. A demonstrably elevated activity of haemolymph lysozyme and trace activity of cecropins found in pupae of Galleria treated with saline W, a salt solution physiological to moths, disappear soon after 36 hr from injection. 5. 5. In P. brassicae, however, sterile insect Ringer can give a varying, if present at all, immune response. 6. 6. A mechanical injury (sterile wounding of insect body) can occasionally induce a similar but much weaker response. 7. 7. The antibacterial activity was drastically reduced in Pieris or completely depressed in most pupae of Galleria when actinomycin D or cycloheximide was given at an early time post-immunization with E. cloacae. 8. 8. It is concluded that the de novo synthesis of ribonucleic acid and immune proteins is required for expression of antibacterial activity in pupal haemolymphs. 9. 9. The synthesis of an immune mRNA was completed about 7 hr after the injection of the immunizing bacteria.

Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa

Pseudomonas aeruginosa K437 lacks the ferripyoverdine receptor and, as a result, grows poorly on an iron-deficient minimal medium supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and pyroverdine. By using a phagemid-based in vivo cloning system, attempts were made to clone the receptor gene by complementing this growth defect. Several recombinant phagemids carrying P. aeruginosa chromosomal DNA which provided for good growth on EDDHA-pyoverdine-containing medium and which concomitantly restored production of the ferripyroverdine receptor in strain K437 were isolated. These phagemids contained a common 4.6-kb SphI fragment which similarly restored production of the receptor in K437. Nucleotide sequencing of the SphI fragment revealed a single large open reading frame, designated fpvA (ferripyoverdine uptake), of 2439 bp. The predicted translation product of fpvA has a molecular mass of 89,395 Da. N-terminal amino acid sequence analysis of the purified ferripyoverdine receptor confirmed fpvA as the receptor gene. Moreover, it indicated that the receptor is initially synthesized as a precursor with a signal sequence of 27 amino acids which is cleaved to yield the mature protein. The deduced FpvA polypeptide exhibited homology to regions shown to be conserved in TonB-dependent receptor proteins. FpvA also shared strong homology (41.3% identity) with the PupA protein of Pseudomonas putida WCS358. This protein is the receptor for the iron-bound form of pseudobactin, a compound structurally very similar to pyoverdine.

Rearing success of a polyphagous predatorGeocoris ochropterus (Hem.: Lygaeidae) on preserved ant pupae ofOecophylla smaragdina

Some of the life-parameters such as nymphal development periods, growth (by weight), food consumption, egg laying period, rate, percent mortality ofGeocoris ochropterus (Fieber) have been studied on ant pupae (Oecophylla smaragdina Fabr.), a potential food available both naturally and commercially. The moisture and basic nutrients including protein, carbohydrate and lipid of the cold-preserved ant pupae have been determined to provide understanding of the dietary value of the food for mass rearing. Experiment with variable male company showed that total fecundity exceeds by a narrow margin in constant male company over fortnightly mating programme. No mating leads to reduction of egg laying in virgins.

Protein synthesis in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus: resistance to viral infection acquired during pupal period.

Protein synthesis has been studied in pupae of the silkworm Bombyx mori (Bm) infected with nuclear polyhedrosis virus (BmNPV) at various stages of the pupal period. Nascent proteins were labelled by injection of [35S]methionine into pupae and then analysed by SDS-PAGE. Temporal regulation of synthesis of infected cell-specific proteins (ICSPs) in pupae was demonstrated by electrophoretic analysis of the proteins labelled at different times post-infection (p.i.). The rate of ICSP synthesis reached a maximum at 4 to 5 days p.i., exceeding the rate of synthesis of cellular proteins in uninfected pupae by about twofold. The viral proteins p10 and polyhedrin were the most abundant products synthesized late in the infection. Both proteins were found to be associated with the nuclear matrix after fractionation of nuclei from infected pupae. Two virus-induced phosphoproteins, pp35 and ppB, were found to be the major acceptors of labelled phosphate from [gamma-32P]ATP during in vitro phosphorylation of proteins in pupal homogenates, nuclei and nuclear extracts. These proteins had electrophoretic mobilities comparable to those of structural phosphoproteins of BmNPV virions with M(r)s of 35K and 11K to 16K, respectively. The latter polypeptide was identified as the major DNA-binding protein of the virus. The susceptibility of silkworms to BmNPV decreased markedly during the pupal period. Following injection of BmNPV all young pupae acquired polyhedrosis and finally died whereas most of the older pupae did not exhibit disease and completed metamorphosis normally. Moreover, the later in the pupal period the silkworms were infected, the lower the production of polyhedrin in diseased pupae.

(B2) A set of genes coding for the inducible antibacterial proteins in cecropia pupae : K.G. Xanthopoulos, Dept. of Microbiology, Univ. of Stockholm, S-106 91 Stockholm, Sweden

(B2) A set of genes coding for the inducible antibacterial proteins in cecropia pupae : K.G. Xanthopoulos, Dept. of Microbiology, Univ. of Stockholm, S-106 91 Stockholm, Sweden

Differential synthesis of bacteria-induced proteins of Manduca sexta larvae and pupae

The range of inducible antibacterial and other associated haemolymph proteins in Manduca sexta larvae and pupae was examined by high resolution two-dimensional (2D) isoelectric focusing-polyacrylamide gel electrophoresis. Twenty-two major inducible proteins were consistently resolved on gels of haemolymph from bacteria-injected larvae. Haemolymph from bacteria-injected pupae showed a different pattern of induced proteins. The proteins of the two stages include those which (i) are induced in both stages, (ii) those which are exclusively induced in either larvae or pupae, (iii) those which are inducible in larvae, but consititutively present in pupae, and, (iv) those which are induced in larvae, and which are present at intermediate levels but may be induced to higher levels in pupae. The antibacterial activity of the haemolymph from larvae and pupae was compared on acidpolyacrylamide gels, and the apparent M r and pI of the inducible proteins determined. Certain of the inducible proteins appear to resemble the cecropin and attacin proteins of Hylophora cecropia.

Effects of ecdysteroid on protein synthesis of Bombyx ovary.

Administration of a high dose (100 μg) of 20-hydroxyecdysone to a Bombyx pupa aged 2 days caused a reduction in content of ribosomes, translatable mRNA, and polysomes of the ovaries. This coincided with the lower relative content of putative chorion proteins in the hormone-treated pupae. However, no large influence of the hormone treatment was detected at the translational level. The developmental time of the ovaries was much prolonged in the hormone-treated animals, and this seemed to compensate for the above decrease in protein content and finally caused a deposition of a normal amount of chorion proteins in oviposited eggs. These results support the idea that the amount of chorion proteins is related to the developmental time of the ovary.

An analysis of major protein species during pupation in Megachile rotundata

Total protein of prepupae, pupae and adults of Megachile rotundata was analyzed by one and two dimensional polyacrylamide gel electrophoresis. Polypeptide 1 with an apparent mol. wt of 107,000 and apparent isoelectric point of 6.55 was the major protein species found in the prepupal stage. Polypeptide 1 decreased during development and was absent in newly emerged adults. The apparent mol. wt and isoelectric point of ten other major proteins was determined.

Disk-elektrophoretische fraktionierung der Hämolymphe-proteine Während der Metamorphose verschiedener Kasten und Geschlechter von Formica pratensis

Using polyacrylamide electrophoresis the proteins of the haemolymph of the different developmental stages can be separated into eight strong and nine weak coloured fractions during the cocoon period of Formica pratensis. The proteins were stained with aniline black and measured quantitatively by a Chromoscan densitometer. The values were compared with those maintained with bovine serum albumin. The total protein content of the haemolymph was calculated as the sum of the different fractions; at maximum it amounts to 2·1 per cent (w/v). The maximum is reached during the pharate pupal stage and during the pigmentation of the eyes; the minimum can be observed at the end of pupal ecdysis. At the beginning of body pigmentation in all the forms the protein content of the haemolymph was very much reduced, especially in workers and females. All fractions change independently resulting in a different composition of the haemolymph proteins in pharate pupae, eclosed pupae, and pharate adults. The slow-running fractions f1, f3, f5, and f6 and the mean bands f8 and f11 are reduced weakly until body pigmentation, and from the eleventh day strongly in both castes. All fractions are reduced during the cocoon period, but mostly the slow-running ones. Only the front band f14 increases to nearly twice that of the protein content. The importance of the changes in the protein fractions for development of different organs and for the synthesis of the haemolymph proteins and the influence of hormones are discussed.