The Synthetic Study of 2.3-Dihydro-lH-pyrrolo [1,2-a] indole Ring System

The 2,3-Dihydro-lH-pyrrolo[l,2-a]indole ring system was synthesised by the condensation reaction of toluquinone and 2-cyanomethylenepyrrolidine.     

Purification and Properties of meso-α,∊-Diaminopimelate Decarboxylase from Bacillus sphaericus

Soybean 7S and 11S globulins were stored at relative humidities (RHs) of 11% and 96% at 50°C. The redispersibility of the proteins at RH 96% decreased in a short time. However, it did not decrease, when stored for 45 days at RH 11%. Gel filtration showed that the proteins polymerized during storage. The effects of urea, sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) on the redispersibilities of the proteins at RH 96% showed that the hydrogen, hydrophobic and disulfide bonds participate in the polymerization of 7S globulin, and that the disulfide bond is strongly related to the polymerization of 11S globulin. Redispersibility was restored with 2-ME in both the 7S and 11S globulins and some of the proteins in the supernatant redispersed with 2-ME were observed to be similar to the native ones with respect to the gel filtration, electrophoretic behavior and circular dichroism spectrum.     

Cloning and Expression of the Pseudomonas Gene for Utilization of d-Leucine in Escherichia coli

Two genes of Pseudomonas putida (IFO 12996) which code for enzymes participating in amino acid metabolism, were cloned in Escherichia coli C600 using pBR322 as a vector. pST7549 is a 7.9 kb hybrid plasmid DNA which is composed of four SalI fragments (0.3, 1.4, 1.9 and 4.3 kb), and codes for β-isopropylmalate dehydrogenase (EC 1.1.1.85) in l-leucine biosynthesis. The enzyme activity in the crude extract from E. coli C600 bearing pST7549 was 80 ~ 90% lower than that of E. coli K12 or P. putida. When the foreign SalI fragments derived from P. putida were subcloned, a 1.9 kb SalI fragment was found to encode β-isopropylmalate dehydrogenase and it did not contain the promoter of P. putida DNA. Plasmid pST6961 has a 1.8 kb insert derived from the P. putida DNA in the SalI site of pBR322. E. coli cells carrying this recombinant plasmid show no leucine racemase activity and no d-leucine transaminase activity, but five-times higher d-leucine oxidation activity than the host strain, E. coli. Enzymological studies have suggested that plasmid pST6961 codes for d-amino acid dehydrogenase, a key enzyme in d-amino acid metabolism.     

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.     

An Improved Synthesis of DL-Canavanine

Excellent l-proline producers were screened for among sulfaguanidine resistant mutants derived from three typical l-glutamic acid-producing bacteria: Brevibacterium flavum, B. lactofermentum, and C. glutamicum.

The best strain, No. 199, is a sulfaguanidine resistant mutant derived from an isoleucine auxotroph of B. flavum 2247 by nitrosoguanidine. Strain No. 199 produced 35 mg/ml of l-proline after 72 hr of cultivation with 10% glucose as a carbon source. The strain also accumulated purine bases such as adenine, guanine, and hypoxanthine, i.e., degradation products of purine nucleotides. In the mutant, 1.6 ~ 2.0 fold more intracellular ATP was found than that in the parent strain; it is a substrate of glutamate kinase relating to l-proline biosynthesis.

On the contrary, the levels of intracellular glutamic acid, a substrate of glutamate kinase, were similar among these strains.

It was confirmed that the increment of internal ATP, which was important in the l-proline production mechanism, was very effective in the improvement of l-proline producers.     

Nematocidal activity of 6-O-octanoyl- and 6-O-octyl-d-allose against larvae of Caenorhabditis elegans

ABSTRACT

The nematocidal activities of the fatty acid esters of d-allose were examined using the larvae of C. elegans. Among the fatty acid esters, 6-O-octanoyl-d-allose (3) showed significant activity. 6-O-octanoyl-d-glucose (5) showed no activity, indicating that the D-allose moiety is essential for the nematocidal activity of 3. A nonhydrolyzable alkoxy analog 6-O-octyl-d-allose (6) also showed activity equivalent to that of 3.     

Elution of dissolved Fe from mangrove soil by tannin solution

To reveal the role of tannins in mangroves, tannins in mangrove leaves and the Fe eluted from mangrove soil by adding tannin solutions of different salinity levels was investigated. Leaves of six mangrove and 16 non-mangrove species, and samples of a mangrove floor, Andosol and dark red soil were collected. Results were: (1) Increasing tannic acid concentration to ~50 mM, increased the Fe eluted from mangrove soil to ~20 μgg^?1. (2) When a 100 mM tannic acid solution was added, the Fe eluted from mangrove soil was 5.5 times higher than dark red soil. (3) Although elution of Fe from mangrove soil was higher than in Andosol one day after submersion in a 10 mM tannic acid solution, the difference was stable after 2 days. (4) The elution of Fe from all soils significantly decreased with increasing salinity of a 10 mM tannic acid solution. However, the amount from mangrove soil was 6.1 times higher than dark red soil even with 35 ‰ salinity. (5) The tannin content in the mangrove leaves was 99 ± 16 mgg^?1 and non-mangrove leaves was 76 ± 19 mgg^?1. (6) The Fe eluted from mangrove soil had a positive correlation with the tannin concentrations in the added leaf solution. Tannins in mangrove species promote the elution of Fe from mangrove floor soil even in saline water. Fe complexes were formed when mangrove soil was mixed with leaf tannins suggesting that Fe produced by tannins in mangrove leaves growing in land/sea interfaces likely plays a direct role in marine ecosystems.     

Impaired Neural Differentiation of Induced Pluripotent Stem Cells Generated from a Mouse Model of Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid storage disease that arises from mutations in the Hexb gene and the resultant deficiency in β-hexosaminidase activity. This deficiency results in aberrant lysosomal accumulation of the ganglioside GM2 and related glycolipids, and progressive deterioration of the central nervous system. Dysfunctional glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. Induced pluripotent stem cell (iPSC) technology offers new opportunities for both elucidation of the pathogenesis of diseases and the development of stem cell-based therapies. Here, we report the generation of disease-specific iPSCs from a mouse model of SD. These mouse model-derived iPSCs (SD-iPSCs) exhibited pluripotent stem cell properties and significant accumulation of GM2 ganglioside. In lineage-directed differentiation studies using the stromal cell-derived inducing activity method, SD-iPSCs showed an impaired ability to differentiate into early stage neural precursors. Moreover, fewer neurons differentiated from neural precursors in SD-iPSCs than in the case of the wild type. Recovery of the Hexb gene in SD-iPSCs improved this impairment of neuronal differentiation. These results provide new insights as to understanding the complex pathogenic mechanisms of SD.