Effects of the surface stoichiometry of seeds on GaN layer growth by hydride vapour phase epitaxy.

The effect of the atmosphere in a reactor prior to hydride vapour phase epitaxy on the surface stoichiometry of both the GaN template and layer growth was studied. The surface stoichiometry of metallic Ga layers was clarified by X-ray photoelectron spectroscopy using templates without NH3 protection. The metallic Ga layer acted as a mask and exerted a significant effect on the subsequent epitaxial layer growth mode. GaN grown on the template without protection followed island growth in the initial growth stage. In contrast, GaN epitaxy on the template with NH3 protection quickly converts to pseudo-2D growth. The images of CL illustrate that the GaN epilayer on the template without protection has a lower dislocation density than the GaN epilayer grown on the template with NH3 protection. Reasons behind this effect have been discussed.

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.

The impacts of classical insect hormones on the expression profiles of a new digestive trypsin-like protease (TLP) from the cotton bollworm, Helicoverpa armigera.

Trypsin proteinases perform important roles in the protein digestion of an insect midgut. A 1042 bp full-length cDNA was cloned from Helicoverpa armigera. The gene encoded a 32 kDa protein, with a predicted isoelectric point of 5.7. The amino acid sequence of the protein had a trypsin-like serine protease domain, and the gene was named Ha-TLP. The expression of the gene was tissue-specific and the transcript of Ha-TLP existed only in the midgut and was not found in the head-thorax, integument, fat body and haemocytes from 5th instar larvae, with similar expression levels between those in feeding larvae and in molting larvae. In the midgut, the gene transcription level declined from 6th instar 72 h after the larvae entered the wandering stage, and disappeared from 6th instar at 96 h until the pupal stage. By immunohistochemistry, Ha-TLP was detected in the cytoplasm of the midgut epithelial cells of the 6th instar feeding stage worms. The expression of Ha-TLP could be up-regulated by a juvenile hormone (JH) analog methoprene and down-regulated by 20-hydroxyecdysone (20E). These facts indicate that Ha-TLP was involved in food digestion during larval growth and probably up-regulated by JH and suppressed by extra 20E in vivo.

Production of recombinant androgen receptor in a heterologous expression system.

To facilitate detailed studies of androgen receptor, we have produced a full-length receptor protein and some of its deletion mutants in Spodoptera frugiperda (Sf9) insect cells, using the baculovirus expression system. Recombinant baculovirus DNA-infected Sf9 cells expressed these proteins in very high quantities, which represented as much as 30-40% of total insect cell protein at 72 h after infection. Only < 1% of the recombinant protein was soluble in low-salt buffers; the majority formed electron-dense cytoplasmic aggregates 30-40 nm in diameter. These aggregates could be solubilized in 6 mol/L guanidine HCl, and biologically active receptor was generated by diluting the guanidine HCl preparation 20- to 50-fold. The full-length receptor, expressed either in a soluble or aggregated form, had characteristics typical of a native receptor: it bound steroids with high affinity and specificity, interacted with DNA in a sequence-specific fashion, and was recognized by domain-specific receptor antibodies. Androgen-receptor protein purified to homogeneity in guanidine HCl required the presence of Zn2+ ions during the refolding to reconstitute its DNA-binding form; ZnCl2 was not, however, needed to restore the receptor's steroid-binding activity.

Expression of androgen receptor in insect cells. Purification of the receptor and renaturation of its steroid- and DNA-binding functions.

A full-length rat androgen receptor cDNA was used to produce a recombinant baculovirus (AcrAR) by homologous recombination. Spodoptera frugiperda (Sf9) cells infected with this virus expressed a 110-kDa polypeptide that amounted up to about one-third of total cell protein. Studies with AR antibodies confirmed that this protein was indeed rAR. Only a minor portion of the recombinant AR was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 M guanidine HCl (GdnHCl). Electron microscopy of cell pellets revealed that AR was localized to electron-dense cytoplasmic aggregates. The soluble cytosolic receptor was biologically active, in that it bound [3H]mibolerone with high affinity and specificity and interacted with an androgen-responsive element. The functions of the GdnHCl-solubilized AR were partially restored by a 20-50-fold dilution. The solubilized receptor was purified to an apparent homogeneity in a single step by gel filtration on a Sephacryl S-400 column in the presence of 6 M GdnHCl. The homogeneous AR protein could be renatured to bind [3H]mibolerone, interact specifically with a DNA element, and be recognized by receptor antibodies. Receptor-DNA interaction was stabilized by an antibody directed against the N-terminal part and abolished by an antibody against the hinge region of the receptor Zn2+ ions were essential for the purified receptor to refold into a specific DNA-binding form during the renaturation, with the optimal ZnCl2 concentration being 50-100 microM depending on the buffer conditions. Cd2+ ions were also capable of restoring the receptor's DNA-binding activity and did so at concentrations 10-fold lower than those of the Zn2+ ions.