Isolation of a Novel Auxin,Methyl 4-Chloroindoleacetate from Immature Seeds of Pisum sativum

Human casein was separated by gel filtration on a column of Sephadex G–200 with 0.1 m Tris buffer (pH 8.5) containing 1.0 m NaCl. The effluent which increased in turbidity at 25°C was centrifuged at 25,000 × g for 30 min and the precipitate was obtained as Fraction 6. After centrifugation, the effluent was separated into 5 elution fractions.

Disc gel electrophoretic patterns of each fraction showed occurrence of secondary bands other than major bands especially in Fractions 3, 4 and 5. The casein solutions unheated and heated at 100°C for 5 and 10 min were kept at 5°C for 5 days. No marked changes of electrophoretic pattern were observed among these casein solutions. However, when a casein solution heated at 100°C for 5 min was chroma to graphed under the same condition, secondary bands also appeared.     

Isolation and Identification of Ubiquinone 10 from Cultured Cells of Tobacco

Callus tissues were induced from stem and root segments of Rauwolfia serpentina. Growth and alkaloid production of the callus tissues were examined under various culture conditions. The growth was strikingly promoted in the presence of 2,4-D (0.5~1 ppm), kinetin (0.2~0.5 ppm) and yeast extract (0.1~0.2%). At favourable conditions, the growth value in 4 weeks’ culture was ca. 40 (F.W.), and ca. 25 (D.W.) for stem callus tissues, and ca. 15 (F.W.), and ca. 8 (D.W.) for root callus tissues. Stem and root callus tissues produced ajmaline and some other unidentified Rauwolfia alkaloids. The ajmaline content in root callus tissues was 10~20mg % and in stem callus tissues was 1~10mg %. The ajmaline production was strikingly reduced when 2,4-D concentration increased, or kinetin was omitted in the culture medium. Phytosterols including stigmasterol, β-sitosterol or cholesterol were also produced.     

Synergistic Taste Effects of Some New Ribonucleotide Derivatives

Taste effects of six newly synthesized ribonucleotide derivatives, i.e., disodium salts of 2-methyl-5′-inosinic acid · 6H₂O, 2-ethyl-5′-inosinic acid · 1.5H₂O, 2-N-methyl-5′-guanylic acid · 5.5H₂O, 2-N-dimethyl-5′-guanylic acid · 2.5H₂O, 2-methylthio-5′-inosinic acid · 6H₂O and 2-ethylthio-5′-inosinic acid · 2H₂O, were studied. Stimulus thresholds (detection thresholds) of these derivatives ranged from about 0.02 to 0.006 g/100ml. Flavor-enhancing activities of them were 2.3 to 8.0 times larger than that of disodium 5′-inosinate · 7.5H₂O IMP) in the synergistic effect with monosodium glutamate. Furthermore, the quality of taste of all the derivatives was recognized to be the same kind to that of IMP.     

Secondary structural analysis of proteins based on 13C chemical shift assignments in unresolved solid-state NMR spectra enhanced by fragmented structure database

Magic-angle-spinning solid-state ¹³C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-¹³C,¹⁵N labeled samples. To overcome this problem, we present a method for assigning ¹³C chemical shifts and secondary structures from unresolved two-dimensional ¹³C–¹³C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to ¹³C^α, ¹³C^β, and ¹³C′ chemical shifts and cross-peak intensities. The experimental ¹³C–¹³C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific C^α, C^β, and C′ chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50–200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved ¹³C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.     

DNA Methylation Profile Distinguishes Clear Cell Sarcoma of the Kidney from Other Pediatric Renal Tumors

A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms’ tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing’s sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms’ tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.     

Epitope Mapping for Monoclonal Antibody Reveals the Activation Mechanism for αVβ3 Integrin

Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs) were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.     

Relationship between G1287A of the NET Gene Polymorphisms and Brain Volume in Major Depressive Disorder: A Voxel-Based MRI Study

Background

Earlier studies implicated norepinephrine transporter (NET) gene (SLC6A2) polymorphisms in the etiology of major depressive disorder (MDD). Recently, two single nucleotide SLC6A2 polymorphisms, G1287A in exon 9 and T-182C in the promoter region, were found to be associated with MDD in different populations. We investigated the relationship between the brain volume and these two polymorphisms of the SLC6A2 in MDD patients.

Methods

We obtained 3D high-resolution T1-weighted images of 30 first-episode MDD patients and 48 age- and sex-matched healthy subjects (HS). All were divided into 4 groups based on polymorphism of either the G1287A or the T-182C genotype. VBM analysis examined the effects of diagnosis, genotype, and genotype-diagnosis interactions.

Results

Diagnosis effects on the brain morphology were found in the left superior temporal cortex. No significant genotype effects were found in the T-182C and the G1287A. A significant genotype (G1287A)–diagnosis interaction was found in the left dorsolateral prefrontal cortex. No significant genotype (T-182C)–diagnosis interaction effects were observed in any brain region.

Conclusions

In MDD patients there seems to be a relationship between the volume of the dorsolateral prefrontal cortex and polymorphism of the SLC6A2 G1287A gene.