Ecdysone metabolism in Pieris brassicae during the feeding last larval instar

Ecdysone metabolism in Pieris brassicae during the feeding last larval stage was investigated by using ³H-labeled ecdysteroid injections followed by high-performance liquid chromatographic (HPLC

1 Abbreviations: 3DE = 3-dehydroecdysone; 3D20E = 3-dehydro-20-hydroxyecdysone; 2026E = 20,26-dihydroxyecdysone; E = ecdysone; Eoic = ecdysonoic acid; 2026E′ = 3-epi-20,26-dihydroxyecdysone; E′ = 3-epiecdysone; E′oic = 3-epiecdysonoic acid; E′8P = 3-epiecdysone 3-phosphate; 20E′ = 3-epi-20-hydroxyecdysone; 20E′3P = 3-epi-20-hydroxyecdysone 3-phosphate; FT = Fourier transform; HPLC = high-performance liquid chromatography; 20E = 20-hydroxyecdysone; 20Eoic = 20-hydroxyecdysonoic acid; NMR = nuclear magnetic resonance; NP-HPLC = normal phase HPLC; RP-HPLC = reverse phase HPLC; TFA = trifluoroacetic acid; Tris = tris(hydroxymethyl)-aminomethane.

) analysis of metabolites. Metabolites were generally identified by comigration with available references in different HPLC systems. Analysis of compounds for which no reference was available required a large-scale preparation and purification for their identification by ¹H nuclear magnetic resonance spectrometry. The metabolic reactions affect the ecdysone molecule at C-3, C-20, and C-26, leading to molecules which are modified at one, two, or three of these positions. At C-20, hydroxylation leads to 20-hydroxyecdysteroids. At C-26, hydroxylation leads to 26-hydroxyecdysteroids which can be further converted into 26-oic derivatives (ecdysonoic acids) by oxidation. At C-3, there are several possibilities: there may be oxidation into 3-dehydroecdysteroids, or epimerization possibly followed by phosphate conjugation. Thus, injected 20-hydroxyecdysone was converted principally into 20-hydroxyecdysonoic acid, 3-dehydro-20-hydroxyecdysone, and 3-epi-20-hydroxyecdysone 3-phosphate. Labelled ecdysone mainly gave the same metabolites doubled by a homologous series lacking the 20-hydroxyl group.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Trimodal GSTT1 and GSTM1 genotyping assay by real-time PCR

The GSTT1 and GSTM1 genes are characterized by the existence of a GST*0 null allele responsible for a lack of enzyme activity, with the respective null genotypes GSTT1*0/0 and GSTM1*0/0. The three resulting genotypes (GSTs*1/1, *1/0 and *0/0) are associated with a trimodal distribution of glutathione-conjugator activity. Previous epidemiological studies have only evaluated the cancer risk associated with the GST null genotype relative to the two GST carrier genotypes (GSTs1*1/1 and *1/0). We developed GSTT1 and GSTM1 TaqMan real-time quantitative PCR assays to discriminate each of the three genotypes, with the albumin gene (ALB) as reference. The mean N(GSTT1*1/1) value was 1.0 (95% confidence interval 0.80-1.20). The mean N(GSTT1*1/0) value was 0.48 (95% CI 0.36-0.60). One (3.4%) of the 29 DNA samples yielded the GSTM1*1/1 genotype (N(GSTM1*1/1) = 1), a frequency in keeping with the Hardy-Weinberg distribution. The mean N(GSTM1*1/0) value was 0.50 (95% CI 0.42-0.58). All GSTT1*0/0 and GSTM1*0/0 samples yielded N(GST) values of 0 (Ct = 40); the frequencies of these genotypes (27.6% and 55.2%, respectively) were in keeping with published data. The GSTT1 and GSTM1 real-time PCR assays described here unambiguously discriminate each of the three existing genotypes which should be valuable for assessing the relative risk of cancer associated with each of the three GST genotypes.     

Solution structure of a peptide derived from the oncogenic protein beta-Catenin in its phosphorylated and nonphosphorylated states

Beta-Catenin plays an essential role in the Wingless/Wnt signaling cascade. Phosphorylation of beta-Catenin in its N-terminal region by the kinase GSK-3beta is required for the interaction with the SCF-beta-TrCP protein complex that targets beta-Catenin for proteasome degradation. In the present work, we used two peptides of 32 amino acids referred to beta-Cat17-48 and P-beta-Cat17-48 for the phosphorylated peptide at the two sites Ser33 and Ser37. Circular dichroism and NMR techniques were used to assess the influence of the phosphorylation. The spectra of the peptides at pH 7.2 were completely assigned. Analysis of the medium-range NOE connectivities indicated that beta-Cat17-48 seems to be only poorly folded. These data are in agreement with the result of structure calculations. P-beta-Cat17-48 possesses two helical segments around the DpSGXXpS motif, which forms a large bent with the phosphate groups pointing out of the structure. On the contrary, beta-Cat17-48 shows less well-defined secondary structures and appears as a more flexible peptide, but adopts in the motif DSGXXS a more compact conformation than P-beta-Cat17-48. Differences in this molecular region suggest that conformational changes of phosphorylated beta-Catenin play an important role for the interaction with the SCF-beta-TrCP protein complex.     

MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation

Background

Irreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.

Methods

Nineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.

Results

MMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).

Conclusion

During exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.     

STD and TRNOESY NMR studies on the conformation of the oncogenic protein beta-catenin containing the phosphorylated motif DpSGXXpS bound to the beta-TrCP protein

beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that beta-TrCP targets the oncogenic protein beta-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from beta-catenin containing the phosphorylated motif DpSGXXpS (P-beta-Cat17-48) and beta-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the beta-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter beta-catenin fragment with the beta-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated beta-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Ozonolysis of 2'-Deoxycytidine: Isolation and Identification of the Main Oxidation Products

The ozone-mediated oxidation of 2'-deoxycytidine (dCyd) was investigated on the basis of final product identification. The oxidation reaction gave rise to five major modified nucleosides which were isolated and characterized on the basis of extensive ¹H NMR and mass spectrometry measurements. The comparison with the current knowledge of the hydroxyl radical mediated oxidation reactions of 2'-deoxycytidine in aerated aqueous solution, indicates that the formation of ozone oxidation products may be mostly explained by the opening of the pyrimidine C₅-C₆ double bond. Thus, the formation of the identified products obtained by ozonolysis of 2'-deoxycytidine is accounted for by the initial generation of an ozonide.