Production enhancement and refolding of caprine growth hormone expressed in Escherichia coli

This study describes comparison between IPTG and lactose induction on expression of caprine growth hormone (cGH), enhancing cell densities of Escherichia coli cultures and refolding the recombinant cGH, produced as inclusion bodies, to biologically active state. 2–3 times higher cell densities were obtained in shake flask cultures when induction was done with lactose showing almost same level of expression as in case of IPTG induction. With lactose induction highest cell densities were achieved in TB (OD₆₀₀ 16.3) and M9NG (OD₆₀₀ 16.1) media, producing 885 and 892 mg cGH per liter of the culture, respectively. Lactose induction done at mid-exponential stage resulted in a higher cell density and thus higher product yield. cGH over-expressed as inclusion bodies was solubilized in 50 mM Tris–Cl buffer (pH 12.5) containing 2 M urea, followed by dilution and lowering the pH in a step-wise manner to obtain the final solution in 50 mM Tris–Cl (pH 9.5). The cGH was purified by Q-Sepharose chromatography followed by gel filtration with a recovery yield of 39% on the basis of total cell proteins. The product thus obtained showed a single band by SDS–PAGE analysis. MALDI-TOF analysis showed a single peak with a mass of 21,851 dalton, which is very close to its calculated molecular weight. A bioassay based on proliferation of Nb2 rat lymphoma cells showed that the purified cGH was biologically active.     

Structural prediction and analysis of VIH-related peptides from selected crustacean species

The tentative elucidation of the 3D-structure of vitellogenesis inhibiting hormone (VIH) peptides is conversely underprivileged by difficulties in gaining enough peptide or protein, diffracting crystals, and numerous extra technical aspects. As a result, no structural information is available for VIH peptide sequences registered in the Genbank. In this situation, it is not surprising that predictive methods have achieved great interest. Here, in this study the molt-inhibiting hormone (MIH) of the kuruma prawn (Marsupenaeus japonicus) is used, to predict the structure of four VIHrelated peptides in the crustacean species. The high similarity of the 3D-structures and the calculated physiochemical characteristics of these peptides suggest a common fold for the entire family.     

A novel solid phase approach to Aia‐containing peptides

A strategy was developed to directly assemble 4‐amino‐1,2,4,5‐tetrahydro‐indolo[2,3‐c]‐azepin‐3‐ones on solid‐phase‐supported peptide sequences. Fmoc‐ and Boc‐based strategies were investigated. The Fmoc‐strategy approach strongly depends on the peptide sequence being synthesized while the Boc‐based synthesis leads to excellent results for all the selected peptide analogs. The method was applied to prepare Aia‐analogs of several bioactive peptides containing one or more Trp‐residues which were shown to be important for biological recognition. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

An insect farnesyl phosphatase homologous to the N-terminal domain of soluble epoxide hydrolase

In insects, farnesyl pyrophosphate (FPP) is converted to juvenile hormone (JH) via a conserved pathway consisting of isoprenoid-derived metabolites. The first step of this pathway is presumed to be hydrolysis of FPP to farnesol in the ring gland. Based on alignment of putative phosphatases from Drosophila melanogaster with the phosphatase domain of soluble epoxide hydrolase, Phos2680 and Phos15739 with conserved phosphatase motifs were identified, cloned and purified. Both D. melanogaster phosphatases hydrolyzed para-nitrophenyl phosphate, however, Phos15739 also hydrolyzed FPP with a K_cat/K_m of 2.1 × 10⁵ M⁻¹ s⁻¹. RT-PCR analysis revealed that Phos15739 was expressed in the ring gland and its expression was correlated with JHIII titer during development of D. melanogaster. N-acetyl-S-geranylgeranyl-l-cysteine was found to be a potent inhibitor of Phos15739 with an IC₅₀ value of 4.4 μM. Thus, our data identify Phos15739 as a FPP phosphatase that likely catalyzes the hydrolysis of FPP to farnesol in D. melanogaster.     

Effects of continuous and pulsatile PTH treatments on rat bone marrow stromal cells

Bone marrow stromal cells (MSCs) differentiation and proliferation are controlled by numerous growth factors and hormones. Continuous parathyroid hormone (PTH) treatment has been shown to decrease osteoblast differentiation, whereas pulsatile PTH increases osteoblast differentiation. However, the effects of PTH treatments on MSCs have not been investigated. This study showed continuous PTH treatment in the presence of dexamethasone (DEX) promoted osteogenic differentiation of rat MSCs in vitro, as demonstrated by increased alkaline phosphatase (ALP) activity, number of ALP expressing cells, and up-regulation of PTH receptor-1, ALP, and osteocalcin mRNA expressions. In contrast, pulsatile PTH treatment was found to suppress osteogenesis of rat MSCs, possibly by promoting the maintenance of undifferentiated cells. Additionally, the observed effects of PTH were strongly dependent on the presence of DEX. MSC proliferation however was not influenced by PTH independent of treatment regimen and presence or absence of DEX. Furthermore, our work raised the possibility that PTH treatment may modulate stem/progenitor cell activity within MSC cultures.     

The Ski protein can inhibit ligand induced RARα and HDAC3 degradation in the retinoic acid signaling pathway

Recent data has implicated the Ski protein as being a physiologically relevant negative regulator of signaling by retinoic acid (RA). The mechanism by which Ski represses RA signaling is unknown. Co-immunoprecipitation and immunofluorescence assay showed that Ski and RARα are in the same complex in both the absence and presence of RA, which makes Ski different from other corepressors. We determined that Ski can stabilize RARα and HDAC3. These results suggest that Ski represses RA signaling by stabilizing corepressor complex.     

Fatty-acid metabolism is involved in stress-resistance mechanisms of Caenorhabditis elegans

Fatty acids are the major components of the phospholipid bilayer and are involved in several functions of cell membrane. We previously reported that fatty-acid metabolism is involved in the regulation of DAF-2/insulin signal in Caenorhabditis elegans. In this study, we investigate the role of fatty-acid metabolism in stress resistance with respect to daf-16 in nematode. We found that fatty-acid metabolism regulates heat, osmotic, and oxidative-stress resistance in C. elegans. RNA interference (RNAi) of fat-6, fat-7, and elo-2 enhanced heat resistance but decreased oxidative-stress tolerance. RNAi of fat-2 strongly increased osmotic-stress resistance, whereas nhr-49-RNAi remarkably reduced osmotic and oxidative-stress tolerance. In daf-16 mutants (mgDf50), RNAi of fat-2 and fat-7 increased viability under osmotic stress, while RNAi of fat-6, fat-7, and elo-2 enhanced heat resistance. Exposure of saturated fatty acids to RNAi worms of fat-1-, fat-7-, and nhr-49 increased osmotic resistance. On the other hand, polyunsaturated fatty acids (PUFAs) reduced osmotic-stress tolerance in fat-2-RNAi worms, whereas PUFAs enhanced it in nhr-49-RNAi worms. Heat-stress resistance in fat-6- and fat-7-RNAi worms was suppressed by oleic acid.These results suggest that stress-resistance mechanisms are regulated by fatty-acid metabolism with or without DAF-16 activity.     

Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration

Hemolin, a member of the immunoglobulin protein superfamily, functions in Lepidoptera as an opsonin in defence against potential pathogens and seems to play a role in tissue morphogenesis. We show that hemolin gene is expressed in several organs of Galleria mellonella larvae, including the nervous system and the silk glands. The expression in the silk glands of the wandering larvae and their isolated abdomens is enhanced within 6 h after an injection of bacteria, lipopolysaccharides, or peptidoglycans. The magnitude of silk gland response to bacterial challenge is similar to that seen in the fat body. A profound rise of hemolin expression without bacterial inoculation occurs in the silk glands of isolated abdomens when they are induced to pupate by a topical application of 20-hydroxyecdysone (20E). The induction of pupation is associated with silk gland programming for disintegration by apoptosis and phagocytosis. Administration of a juvenile hormone agonist prevents pupation and abolishes the stimulatory 20E effect on the hemolin expression. Hemolin protein can be immunodetected in the silk glands as well as in the spun-out cocoon silk. The results suggest that silk glands are a component of the insect immune system and that hemolin may mark the apoptic cells for the elimination by hemocytes.     

Larval feeding substrate and species significantly influence the effect of a juvenile hormone analog on sexual development/performance in four tropical tephritid flies

The juvenile hormone (JH) analog methoprene reduces the amount of time it takes laboratory-reared Anastrepha suspensa (Caribbean fruit fly) males to reach sexual maturity by almost half. Here, we examined if methoprene exerted a similar effect on four other tropical Anastrepha species (Anastrepha ludens, Anastrepha obliqua, Anastrepha serpentina and Anastrepha striata) reared on natural hosts and exhibiting contrasting life histories. In the case of A. ludens, we worked with two populations that derived from Casimiroa greggii (ancestral host, larvae feed on seeds) and Citrus paradisi (exotic host, larvae feed on pulp). We found that the effects of methoprene, when they occurred, varied according to species and, in the case of A. ludens, according to larval host. For example, in the case of the two A. ludens populations the effect of methoprene on first appearance of male calling behavior and number of copulations was only apparent in flies derived from C. greggii. In contrast, males derived from C. paradisi called and mated almost twice as often and females started to lay eggs almost 1 day earlier than individuals derived from C. greggii, but in this case there was no significant effect of treatment (methoprene) only a significant host effect. There were also significant host and host by treatment interactions with respect to egg clutch size. A. ludens females derived from C. paradisi laid significantly more eggs per clutch and total number of eggs than females derived from C. greggii. With respect to the multiple species comparisons, the treatment effect was consistent for A. ludens, occasional in A. serpentina (e.g., calling by males, clutch size), and not apparent in the cases of A. obliqua and A. striata. Interestingly, with respect to clutch size, in the cases of A. ludens and A. serpentina, the treatment effect followed opposite directions: positive in the case of A. ludens and negative in the case of A. serpentina. We center our discussion on two hypotheses (differential physiology and larval-food), and also interpret our results in light of the life history differences exhibited by the different species we compared.