Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.     

Novel functional biodegradable polymer II: fibroblast growth factor-2 activities of poly(gamma-glutamic acid)-sulfonate

Basic fibroblast growth factor (FGF-2) mitogenic activities of sulfonated poly(gamma-glutamic acid) (gamma-PGA-S) were investigated with chlorate-treated L929 fibroblast culture tests. When 72% of the carboxyl groups in gamma-PGA were sulfonated (gamma-PGA-S72), cell numbers reached a maximum. The activity of gamma-PGA-S72 was higher than that of gamma-PGA and synthetic heparinoids and was almost comparable to that of heparin. Cytotoxicity of gamma-PGA-S72 was not observed, regardless of the degree of sulfonation. FGF-2-protective effects of gamma-PGA-S72 against acid and thermal inactivation were also evaluated, and gamma-PGA-S72 showed higher FGF-2-protective effects in comparison to nonsulfonated gamma-PGA. The steric structures of various sulfonated gamma-PGA-Ss were analyzed by molecular modeling (molecular orbital method (MOPAC)) and indicated that gamma-PGA-Ss are helical in vacuo. Results from MOPAC and the molecular mechanics method (MM2) demonstrated that electrostatic interactions can take place between sulfonic and carboxyl groups of gamma-PGA-S and basic amino acid residues in FGF-2. gamma-PGA-S72 can interact with FGF-2 strongly.     

Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.     

Synthesis and properties of bifunctional chloroalkyl nitrosamines with an intercalating moiety

Three N-nitroso-N-(arylcarbonyloxymethyl)-3-chloropropylamines were synthesized, and their chemical and biological properties were studied. All arylcarboxylates intercalated with double-stranded DNA, and their mutagenicity and DNA cross-linking activity were affected by their ring structure. The DNA interstrand cross-link formation increased dose dependently after treatment with the acridine analog. The anthraquinone analog showed the highest bacterial mutagenicity among the three nitrosamines in Salmonella typhimurium TA100, while in Salmonella typhimurium TA92, which can detect cross-linking agents, the acridine analog showed the highest mutagenicity. This agreed with the result of a cross-linking assay. These results suggest that the three-ring aromatic moiety gives DNA-intercalating ability to cross-linkable chloropropyl nitrosamine, and the acridine analog is considered as a possible new antitumor lead compound.     

Humoral Immune Responses to EGFR-Derived Peptides Predict Progression-Free and Overall Survival of Non-Small Cell Lung Cancer Patients Receiving Gefitinib

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with clinical response to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, in patients with non-small cell lung cancer (NSCLC). However, humoral immune responses to EGFR in NSCLC patients have not been well studied. In this study, we investigated the clinical significance of immunoglobulin G (IgG) responses to EGFR-derived peptides in NSCLC patients receiving gefitinib. Plasma IgG titers to each of 60 different EGFR-derived 20-mer peptides were measured by the Luminex system in 42 NSCLC patients receiving gefitinib therapy. The relationships between the peptide-specific IgG titers and presence of EGFR mutations or patient survival were evaluated statistically.IgG titers against the egfr_481–500, egfr_721–740, and egfr_741–760 peptides were significantly higher in patients with exon 21 mutation than in those without it. On the other hand, IgG titers against the egfr_841–860 and egfr_1001–1020 peptides were significantly lower and higher, respectively, in patients with deletion in exon 19. Multivariate Cox regression analysis showed that IgG responses to egfr_41_ 60, egfr_61_80 and egfr_481_500 were significantly prognostic for progression-free survival independent of other clinicopathological characteristics, whereas those to the egfr_41_60 and egfr_481_500 peptides were significantly prognostic for overall survival. Detection of IgG responses to EGFR-derived peptides may be a promising method for prognostication of NSCLC patients receiving gefitinib. Our results may provide new insight for better understanding of humoral responses to EGFR in NSCLC patients.     

Pyramiding of male-sterile genes in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don with the aid of closely linked markers

Gene pyramiding is a breeding method used to combine multiple useful genes. Although several genes have been pyramided in certain crops, gene pyramiding has not previously been applied to forest trees. In this study, we used the markers closely linked to the two male-sterile genes MS1 and MS2 for the effective development of individuals doubly heterozygous for these two genes. This is the first example of gene pyramiding through marker-assisted selection (MAS) in forest trees. The markers gSNP06239, which is closely linked to the MS1 gene, and estSNP00695, which is closely linked to MS2, were used in MAS. On the basis of the linkage phase between the markers and male-sterile loci, we selected five F₁ individuals (S3-64 from Shindai-3 × Kamikiri-31, S3-70 from Shindai-3 × Kamikiri-38, S3-77 from Shindai-3 × Kamikiri-47, S1-22 from Shindai-1 × Nakakubiki-4, and S1-56 from Shindai-1 × Setsugai-20) as parents for artificial crossing. The 268 seedlings obtained from six artificial cross combinations were used in this study. Chi-squared tests showed no significant deviation from the expected Mendelian ratios of genotypes, indicating that MAS using markers closely linked to the male-sterile genes worked very well. Fifteen individuals that showed unexpected genotypes were probably recombinants, because the map distances between the male-sterile locus and the DNA markers were 4.1 cM (gSNP06239 to MS1) and 6.9 cM (estSNP00695 to MS2). Development of markers more closely linked to the male-sterile loci will facilitate precise gene pyramiding in the future.