Junru Wang

Molecular cloning and characterization of the cathepsin B-like proteinase from the cotton boll worm, Helicoverpa armigera

An enzyme purified from the ovaries of Helicoverpa armigera, as an active form with molecular mass of 30 kDa on SDS‐PAGE, was identified as a cysteine proteinase because it could be inhibited by E‐64, a specific inhibitor of cysteine proteinase, and required reducing conditions for activity. This enzyme was further identified as a cathepsin B‐like cysteine proteinase by partial amino acid sequencing. A cDNA encoding this proteinase was cloned from H. armigera, using degenerate primers and RACE techniques. Results of Northern blots indicated that the mRNA encoding the proteinase was transcribed in the ovaries, the fat bodies of female and male adults, pupae and in the larvae. No mRNA was detected from the larval epidermis or from the midgut. Hence, transcription of the cathepsin B‐like cysteine proteinase from H. armigera was tissue‐specific, but not gender‐ or developmental stage‐specific. However, proteolytic activities were only detected from ovaries, and adult female and male fat bodies. No activity was observed from pupal and larval fat bodies, from the larval epidermis or from the midgut. Only one form of mRNA of ≈ 1100 bases was detected, and in situ hybridization showed that the transcripts were distributed in the adult female fat bodies, follicular cells and the oocytes. Since the proteinase expressed in ovaries was able to degrade vitellin in vitro, it may be involved in the degradation of vitellin during embryonic development.

A cathepsin L-like proteinase is involved in moulting and metamorphosis in Helicoverpa armigera.

Holometabolous insects undergo larval moulting and metamorphosis within their life cycle. A cDNA encoding the cathepsin L‐like proteinase Ha‐cathL has been cloned from Helicoverpa armigera. It has a sequence of 1826 bp and encodes a 550‐residue protein with a molecular mass of 63 kDa. Northern blot analysis indicated that Ha‐cathL is specifically expressed in haemocytes, with increased expression during larval moulting and metamorphosis. In vivo experimentation revealed that Ha‐cathL is up‐regulated by 20‐hydroxyecdysone. Meanwhile, in situ hybridization and immunocytochemistry revealed that Ha‐cathL mRNA is mainly expressed in granulocytes and plasmatocytes. Knock down of cathepsin L by RNA interference results in larvae death before pupation or the formation of a chimeric pupa containing a larval head and thorax, abnormal wings and the pupal abdomen. The reason for this is that the affected haemocytes cannot become granulated, and therefore cannot participate in fat body remodelling and wing development. These facts suggest that Ha‐cathL is involved in larval moulting and metamorphosis by participating in the functioning of haemocytes.

Molecular cloning and expression patterns of the molt-regulating transcription factor HHR3 from Helicoverpa armigera.

Molt‐regulating transcription factors, hormone receptor 3 (HR3), play important roles in regulating expression of tissue‐specific genes involved in insect molting and metamorphosis. A 1668 bp cDNA encoding a molt‐regulating transcription factor (HHR3) was cloned from Helicoverpa armigera, which encodes a protein made up of 556 amino acids. This 62 kDa protein was found to have an isoelectric point (pI) of 6.52. There was no signal peptide or N‐glycosylation site found in this cDNA. A DNA‐binding region signature of nuclear hormone receptor was found from amino acids 107–133. A possible outside to inside transmembrane helice was found from amino acids 72–90. Northern blots of the larvae revealed five bands of HHR3 named as band 0, 1, 2, 3 and 4 with molecular masses determined as 2.1, 2.6, 3.6, 4.5 and 5.5 kb, respectively. The expression patterns of HHR3 in vivo were variable with developmental stages and tissues. Results showed that band 1–4 of HHR3 was only briefly expressed during molting, which suggested these bands are involved in the regulation of molting cascade, whereas band 0 was expressed in both molting and feeding larvae. Band 1 and 2 of HHR3 could be induced from epidermis of newly molted 6th instar larvae by non‐steroidal ecdysone agonist, RH‐2485.

Characterization and influences of classical insect hormones on the expression profiles of a molting carboxypeptidase A from the cotton bollworm (Helicoverpa armigera)

Molting is a very important physiological behavior to arthropods. During molting, integument apolysis occurs, which is the digestion and absorption of the old endocuticle for new cuticle formation. Proteases play critical roles in this process. Molting carboxypeptidase A (Ha‐CPA) is characterized from Helicoverpa armigera. The Ha‐CPA transcript was mainly present in the integument from the 5th instar larvae. In the integument, the transcription level of the gene reached its peak at the 5th instar molting stage and the 6th instar prepupal stage, respectively. The examination of immunohistochemistry revealed that Ha‐CPA could distribute into the molting fluid in the molting‐ and prepupal‐stage larvae. The expression of Ha‐CPA could be up‐regulated by 20‐hydroxyecdysone (20E). These facts indicate that Ha‐CPA participates in the apolysis of the integument during larval molting and metamorphosis.

β-thymosin is upregulated by the steroid hormone 20-hydroxyecdysone and microorganisms

Thymosins have diverse biological activities including actin‐sequestering and tissue repair in vertebrates, however, there is little information about the function of thymosins in invertebrates. We isolated a β‐thymosin gene in Helicoverpa armigera. It has two transcript variants, HaTHY1 and HaTHY2, encoding 19.0 kDa and 14.5 kDa peptides, respectively. HaTHY1 was mainly transcribed in the integument and midgut, while HaTHY2 was principally presented in the fat body and haemocytes. The transcript levels of HaTHY2 showed some fluctuation; there was an obvious increase at the metamorphic stage in the integument or fat body. HaTHY was able to be upregulated by 20‐hydroxyecdysone or by bacterial and viral challenge. These data suggest that HaTHY is upregulated by the steroid hormone and by responses to microorganism infection.

Cu3N and its analogs: a new class of electrodes for lithium ion batteries

Electrode materials with low diffusion energy barriers and high storage capacity of lithium are crucial for high performance rechargeable lithium-ion batteries (LIBs). Based on first-principles calculations, we demonstrate a new class of electrode materials. Taking advantage of the large voids in Cu3N crystals, high lithium mobility and storage capacity can be achieved. The diffusion of Li on Cu3N nanosheets experiences an energy barrier of about 0.09 eV, which is much lower than those of presently proposed electrode materials. The maximum Li capacity of Cu3N nanosheets can reach 1008 mA h g−1. In view of a large number of crystals sharing the same lattice structure as Cu3N, this work opens an avenue for developing electrode materials for high performance LIBs.

Heat shock protein 90 maintains the stability and function of transcription factor Broad Z7 by interacting with its Broad-Complex-Tramtrack-Bric-a-brac domain

Heat shock protein 90 (Hsp90) is a highly conserved chaperone protein that interacts with various client proteins to mediate their folding and stability. The Broad‐Complex‐Tramtrack‐Bric‐a‐brac (BTB) domain, also known as poxvirus and zinc finger (POZ) domain, exists widely in different proteins and is highly conserved. However, the stability mechanism of BTB domain‐containing proteins has not been fully understood. Co‐immunoprecipitation and a protein pull‐down assay were performed to investigate the interaction between Hsp90 and the transcription factor Broad isoform Z7 (BrZ7) in vivo and in vitro. The middle domain of Hsp90 directly associated with the BTB domain of BrZ7. The Hsp90 inhibitor 17‐(Allylamino)‐17‐demethoxygeldanamycin (17‐AAG) interrupted the interaction between Hsp90 and BrZ7 and decreased the protein level of BrZ7 but did not affect the mRNA level of BrZ7. The addition of the proteasome inhibitor peptide aldehyde Cbz‐leu‐leu leucinal suppressed the 17‐AAG‐induced degradation of BrZ7. BTB domain deletion and 17‐AAG treatment resulted in inhibition of BrZ7 function in gene expression in the 20‐hydroxyecdysone and juvenile hormone pathways. These results reveal that the middle domain of Hsp90 associates with the BTB domain of BrZ7 to prevent BrZ7 degradation and maintain BrZ7 function in gene expression in the lepidopteran insect Helicoverpa armigera.

Half-metallic TiF3: a potential anode material for Li-ion spin batteries

As a key component of spintronic devices, spin batteries that can generate spin-polarized current are drawing increasing interests. Herein, we propose a simple strategy for spin batteries by introducing a half-metallic anode material in the conventional Li-ion batteries. Using first-principles calculations, we demonstrate a potential half-metallic anode material, TiF3 crystal, which has stable ferromagnetism and half-metallicity under Li insertion. Low Li diffusion barriers (0.16–0.37 eV) and moderate Li storage capacity (256 mA h g−1) are revealed in the TiF3 crystal. The combination of half-metals and Li-ion battery offers a new solution for spin batteries.

Function of a TGF‐β inducible nuclear protein in the silk gland in Bombyx mori

A TGF‐β inducible nuclear protein 1 (BmTINP1) was cloned from silkworm, Bombyx mori. Polyclonal antibodies against BmTINP1 were produced and subsequently used in immunoblotting and immunohistochemistry analyses. The immunoblotting analyses demonstrated that BmTINP1 was specifically expressed in the anterior silk gland (ASG) and the middle silk gland (MSG) but not in the posterior silk gland (PSG). There were two bands that suggested the existence of an isoform of BmTINP1. The expression profiles of BmTINP1 in ASGs and MSGs were similar, and they manifested a high level of expression throughout the period during which silk gland grew exponentially. Immunohistochemistry results revealed that BmTINP1 was translocated from the nucleus into the cytoplasm when larvae developed from the 4th‐HCS into the 5th instar. 20‐hydroxyecdysone (20E) promotes the translocation, while the methoprene [a juvenile hormone (JH) analog] restrains the process. Our findings indicate that BmTINP1 is involved in silk produce along with the rapid growth of ASGs and MSGs during the last instar larvae, and the process could be regulated by hormones via control of BmTINP1 translocation from the nucleus to the cytoplasm.