Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.     

Influence of the length of the phosphate chain in mRNA 5' cap analogues on their interaction with eukaryotic initiation factor 4E

The recognition of the 5'mRNA cap structure m7G(5')ppp(5')N by one of the components of the initiation translation machinery, the eIF4E factor, plays a pivotal role in regulation of the protein synthesis. In the present study we have shown two opposing roles of the cap phosphate chain in the specific eIF4E-cap interaction. The extension of the phosphate chain enhances the binding of the cap to the unphosphorylated eIF4E but destabilises the eIF4E-cap complex in case of the phosphorylated protein.     

Binding studies of eukaryotic initiation factor eIF4E with novel mRNA dinucleotide cap analogues

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2' of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the "anti reversed" cap analogues, possessing the analogous modifications at C3'. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.     

Interaction between yeast eukaryotic initiation factor eIF4E and mRNA 5' cap analogues differs from that for murine eIF4E

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (Kas) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5' cap than those of the murine and human proteins. The observed differences in the Kas values and the enthalpy changes of the association (deltaH(o)) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.     

Thermodynamics of 7-methylguanosine cation stacking with tryptophan upon mRNA 5' cap binding to translation factor eIF4E

All eukaryotic nuclear transcribed mRNAs possess the cap structure, consisting of 7-methylguanosine linked by the 5'-5' triphosphate bridge to the first nucleoside. The goal of the present study is to dissect the enthalpy and entropy changes of association of the mRNA 5' cap with eIF4E into contributions originating from the interaction of 7-methylguanosine with tryptophan. The model results are discussed in the context of the thermodynamic parameters for the association of eIF4E with synthetic cap analogues.     

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500pm (45%) and 5nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.     

Contrasting effects of VEGF gene disruption in embryonic stem cell-derived versus oncogene-induced tumors

Previous gene targeting studies have implicated an indispensable role of vascular endothelial growth factor (VEGF) in tumor angiogenesis, particularly in tumors of embryonal or endocrine origin. In contrast, we report here that transformation of VEGF-deficient adult fibroblasts (MDF528) with ras or neu oncogenes gives rise to highly tumorigenic and angiogenic fibrosarcomas. These aggressive VEGF-null tumors (528ras, 528neu) originated from VEGF(-/-) embryonic stem cells, which themselves were tumorigenically deficient. We also report that VEGF production by tumor stroma has a modest role in oncogene-driven tumor angiogenesis. Both ras and neu oncogenes down-regulated at least two endogenous inhibitors of angiogenesis [pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP-1)]. This is functionally important as administration of an antiangiogenic TSP-1 peptide (ABT-526) markedly inhibited growth of VEGF(-/-) tumors, with some ingress of pericytes. These results provide the first definitive genetic demonstration of the dispensability of tumor cell-derived VEGF in certain cases of 'adult' tumor angiogenesis, and thus highlight the importance of considering VEGF-independent as well as VEGF-dependent pathways when attempting to block this process pharmacologically.     

The development of new triazole based inhibitors of tumor necrosis factor-alpha (TNF-alpha) production

4-Aryl-5-pyridyl and 4-aryl-5-pyrimidyl based inhibitors of TNF-alpha production, which contain a novel triazole 5-member heterocyclic core, are described. Many pyridyl triazoles containing either an alkyl ether or a substituted aryl side chain on the triazole core showed sub-micromolar activity against LPS-induced TNF-alpha, while pyrimidyl triazoles containing an ethoxymethyl side chain exhibited even better inhibitory activity. Secondary screening data are presented for the pyrimidyl triazoles. Triazole 14e combined excellent potency with good oral bioavailability in the rat.     

Changes in serum copper level during detoxification of acutely poisoned drug addicts

Although it is known that drug addicts are a high-risk group for disruption of many homeostatic processes, little is know about changes in serum trace elements concentrations after taking the psychoactive substances. The aim of the study was to check the influence of the taking homemade heroin on serum level of copper. Blood samples were taken from 30 opiate addicts, and copper concentrations were measured by the means of atomic absorption spectrophotometry. The result of the study show that in the examined group, copper serum concentrations (1.35 mg/L) upon admission to the clinic were higher than in the control group (1.11 mg/L) but decreased during hospitalization (1.18 mg/L). There was no correlation between duration of stay at the hospital and changes in serum copper concentration.     

Differential expression of thrombospondin,collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[¹²⁵l] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450–480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [¹³¹l]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450–480 resolved to Mr 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin l. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from Mrs 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factor 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium. © 1994 Wiley-Liss, Inc.