Role of the androgen receptor in male sexual differentiation.

The two androgens responsible for all aspects of male sexual differentiation are testosterone and dihydrotestosterone. The action of both these steroids is mediated by a specific intracellular receptor, the androgen receptor, which is a member of the nuclear receptor superfamily. The androgen receptor gene has been cloned and is located on the X chromosome at Xq11-12. Mutations of this gene have been found in subjects with both complete and partial androgen insensitivity. In a study of 27 subjects with the androgen insensitivity syndrome, we have identified mutations in 14, using a rapid mutation screening assay. The same technique has also been used to determine carrier status in an affected family. We have also identified a mutation in two brothers who show perineal hypospadias as the only evidence of undervirilisation. Familial severe hypospadias should therefore be included as part of the phenotypic spectrum of partial androgen insensitivity. The study of naturally occurring mutations of the androgen receptor gene is providing further information on the function of the androgen receptor and its role in normal male sexual differentiation.     

Ligand-release pathways in the pheromone-binding protein of Bombyx mori

Pheromone-binding proteins (PBP) supply olfactory neuron cells with pheromones by binding the ligands they are tailored for and carrying them to their receptor. The function of a PBP as an efficient carrier requires fast ligand uptake and release. The molecular basis of the ligand-binding mechanism was addressed here for the intriguing case of the PBP of the silk moth Bombyx mori. This PBP completely encapsulates its ligand bombykol without displaying any obvious ligand entrance/exit sites. Here, two opposite dissociation routes were identified as the most likely entrance/exit paths by replica-exchange molecular dynamics, essential dynamics, and force-probe molecular dynamics simulations. One of the paths runs along a flexible front lid; the other along the termini at the back. Calculated forces and energies suggest that both routes are physiologically relevant. The multiplicity of pathways may reduce or tune the entropic barrier for ligand binding.     

Bombyx mori prohemocyte division and differentiation in individual microcultures

We followed the fate of microcultured Bombyx mori prohemocytes in vitro. Prohemocytes isolated from larval hemolymph (day 1 of 4th instar) were maintained for 4-11 days in serum-free MGM-450 medium and some of them underwent mitotic division. Over 60% of the non-dividing prohemocytes differentiated to plasmatocytes or granulocytes. Some of the granulocytes subsequently transformed to spherulocytes. Of the dividing prohemocytes, 59.2% of the daughter cells differentiated into other types of hemocytes such as plasmatocytes, granulocytes and spherulocytes, and the remainder divided into new prohemocytes. Four of these renewed prohemocytes generated daughter cells composed of plasmatocytes and granulocytes. These results suggest that prohemocytes possess the properties of stem cells, and that plasmatocytes and spherulocytes may be terminally differentiated cells, whereas granulocytes, at least in part, may be a transient form of spherulocyte. Oenocytoids were not produced, suggesting that the lineage of oenocytoids differs from that of other types of hemocytes and that it is determined before release from hemopoietic organs.     

Starvation-responsive glycine-rich protein gene in the silkworm Bombyx mori

Four glycine-rich protein (GRP) genes were identified from expressed sequence tags of the maxillary galea of the silkworm. All four genes were expressed in the maxillary pulp, antenna, labrum, and labium, but none of the genes were expressed in most internal organs. Expression of one of the genes, termed bmSIGRP, was further increased approximately fivefold in the mouth region (including the maxilla, antenna, labrum, labium, and mandible) after 24 h of starvation. bmSIGRP expression peaked at 24 h and gradually declined during the subsequent 2 days. When a synthetic diet not containing proteins was fed, bmSIGRP expression increased significantly in the mouth region to levels similar to that observed in starved larvae. Synthetic diets that lacked vitamins or salts but contained amino acids did not significantly affect bmSIGRP expression. These results suggest that amino acid depletion increases bmSIGRP expression.     

Duality monomer-dimer of the pheromone-binding protein from Bombyx mori

The analysis of a recombinant pheromone-binding protein from the silkworm moth, Bombyx mori, by native gel electrophoresis with Coomassie staining showed one single band with a molecular mass consistent with a monomer. A slow migrating band, detected in the recombinant and native samples by a polyclonal antibody, was indistinguishable from the monomer in the mass spectrum fragmentation pattern and chromatographic behavior. Flow injection analyses of the protein by mass spectrometry in the negative mode showed fragments of a dimer. The dimeric form was also supported by estimation of the molecular mass by gel filtration at basic pH. A cross-linked dimer coeluted with the noncovalent dimer on a gel filtration column. The molecular mass of the protein changed in a pH-dependent way with a dramatic transition from dimer to monomer between pH 6 and 4.5. A low pH induced not only dissociation of the dimer, but also a conformational change in the protein. In marked contrast to denaturation with guanidinium chloride, the emission maxima of tryptophan was not significantly changed at low pH. BmPBP is thus a dimer at slightly acid, neutral, and basic pH, which dissociates and then undergoes conformational change at low pH.     

Pheromone discrimination by the pheromone-binding protein of Bombyx mori

Pheromone-binding proteins are postulated to contribute to the exquisite specificity of the insect's olfactory system, acting as a filter by preferentially binding only one of the components of the natural pheromone. Here, we investigated the possible discrimination of the two very similar components of the natural pheromone gland from the silk moth, Bombyx mori, bombykol and bombykal, by the only pheromone-binding protein (BmorPBP) known to be expressed in the pheromone-detecting sensilla. Free-energy calculations and virtual docking indicate that both bombykol and bombykal bind to BmorPBP with similar affinity. In addition, in vitro competitive binding assays showed that both bombykol and bombykal were bound by BmorPBP with nearly the same high affinity. While BmorPBP might filter out other physiologically irrelevant compounds hitting the sensillar lymph, discrimination between the natural pheromone compounds must be achieved by molecular interactions with their cognate receptors.     

Structure of Bombyx mori chemosensory protein 1 in solution

Chemosensory Proteins (CSPs) represent a family of conserved proteins found in insects that may be involved in chemosensory functions. BmorCSP1 is expressed mainly in antennae and legs of the silkworm moth Bombyx mori and was cloned from antennal cDNA. Here we report the determination of the structure of Bombyx mori CSP1 (BmorCSP1) by NMR. The overall fold of BmorCSP1 is globular and comprises six alpha-helices. These helices span residues 10-14, 17-27, 35-49, 57-72, 75-85, and 92-100. The internal hydrophobic sides of the helices are formed mostly by leucine and isoleucine residues and, therefore, well suited to constitute a binding site for hydrophobic ligands.     

Phylogenetic relationship of Bombyx mori protein disulfide isomerase

A cDNA that encodes protein disulfide isomerase was isolated from Bombyx mori (bPDI), in which an open reading frame of 494 amino acids contained two PDI-typical thioredoxin active sites of WCGHCK and an ER retention signal of the KDEL motif at its C-terminal. The bPDI protein shared less than 55% of the amino acid sequence homology with other reported PDIs. bPDI is most genetically similar to the D. melanogaster PDI. The most serious evolutional diversity was observed between the metazoa and nematoda through PDI evolutional processing. Although bPDI shows a relatively low amino acid homology with other PDIs, in which both sites of the two thioredoxin active sites and the endoplasmic reticulum (ER) retention signal are completely conserved, it was successfully recognized by anti-rat PDI antibodies. This suggests that bPDI may have the activity of a protein isomerase and a chaperone.     

Conformation transition kinetics of Bombyx mori silk protein

Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber.     

Characterization of Rab-interacting lysosomal protein in the brain of Bombyx mori

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori (B. mori), and expressed in Escherichia coli (E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion.     

Stimulated synthesis of the female-specific storage protein in male larvae of the silkworm Bombyx mori treated with juvenile hormone analog

Application of methoprene to fourth (penultimate) instar larvae of the silkworm Bombyx mori induced the appearance of the feeding dauer larvae at the fifth (last) instar and prevented pupal metamorphosis. Methoprene also increased the protein concentrations of hemolymph last instar larvae by preventing sequestration of storage proteins by the fat body. Usually, the female-specific storage protein 1 (SP1)* disappears from the male hemolymph at the time of the last larval instar. However, exposure of male larvae to methoprene at the penultimate instar enhanced the accumulation of SP1 in the hemolymph. The SP1 accumulated in males did not differ in molecular weight and immunoreactivity from the SP1 produced in female larvae. Both sexes of fourth instar larvae allatectomized on day 1 instantly accumulated SP1 in the hemolymph, and methoprene application after allatectomy suppressed the hemolymph accumulation of the SP1. In contrast, if allatectomy was carried out at a later stage of the fourth larval instar, SP1 concentration in hemolymph of fifth instar larvae did not increase, suggesting the different juvenile hormone action for regulation of SP1 synthesis in the penultimate instar larvae of silkworms.     

Lysine acetylation stabilizes SP2 protein in the silkworm Bombyx mori

Lysine acetylation (Kac) is a vital post-translational modification that plays an important role in many cellular processes in organisms. In the present study, the nutrient storage proteins in hemolymph were first found to be highly acetylated—particularly SP2 protein, which contains 20 potential Kac sites. Further results confirmed that lysine acetylation could stabilize and up-regulate the protein level of anti-apoptosis protein SP2, thereby improving the survival of H₂O₂-treated BmN cells and suppressing the apoptosis induced by H₂O₂. The potential mechanism involved in the inhibition of ubiquitin-mediated proteasomal degradation by crosstalk between lysine acetylation and ubiquitination. Our results showed that the increase in the acetylation level by TSA could decrease the ubiquitination and improve the protein level of SP2, indicating that lysine acetylation could influence the SP2 protein level through competition between ubiquitination and the suppression of ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. SP2 is a major nutrient storage protein from hemolymph for amino acid storage and utilization. The crosstalk between lysine acetylation and ubiquitination of SP2 might imply an important role of lysine acetylation for nutrient storage and utilization in silkworm.     

家蚕雄蛾触角电位并联等效电路模型的探讨

【目的】为探讨昆虫嗅觉感受机理及其化学感受行为机制提供技术基础。【方法】对家蚕Bombyx mori雄蛾触角部分切除后,测定其触角电位(EAG)变化;构建家蚕雄蛾并联等效电路模型;分析EAG与电路模拟器件间的关系。【结果】切除部分分枝后,EAG幅值与整根触角相比,除保留中部10个分枝的处理组无显著差别外,其余6组均显著减小;噪声强度有随触角分枝数量减少而增加的趋势;信噪比(SNR)有随分枝数量减少而减小的趋势;触角中部分枝数量由10个逐渐减少时,其EAG幅值随之线性减小。切除尖部主干后,EAG幅值增加,噪声强度减小,SNR增强;切除基部主干后,EAG幅值减小,噪声强度增加,SNR减弱。构建获得了基于触角主干和分枝阻抗的EAG并联等效电路模型,能较好地模拟实验测定结果。【结论】触角主干和分枝的阻抗对EAG信号输出有较大影响,EAG并联等效模型经具体化和改进后可适用于其他类型昆虫触角的EAG实验分析。     

Dynamic conformational equilibria in the physiological function of the Bombyx mori pheromone-binding protein

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP^B), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP^A). This helix α7 is formed by the C-terminal dodecapeptide 131-142, which is flexibly disordered on the protein surface in BmorPBP^B and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1-128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP^B. At pH 4.5, BmorPBP(1-128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of ¹⁵N-¹H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.     

An immune-induced reeler protein is involved in the Bombyx mori melanization cascade

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.