Functional Analysis of Lysosomes During Mouse Preimplantation Embryo Development

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.     

Isolation and enzymatic formation of lignans ofDaphne genkwa andDaphne odora

Four lignans — pinoresinol, lariciresinol, secoisolariciresinol, matairesinol — were isolated from each ofDaphne odora andDaphne genkwa (Thymelaeaceae). Matairesinol isolated from both plants was optically pure (>99% e.e.) and dextrorotatory. Pinoresinol and lariciresinol isolated from the plants were not optically pure, and their enantiomeric compositions ranged from 88% to 95% e.e. in favor of (–)-enantiomers. As for secoisolariciresinol, the one fromD. odora was optically pure [(+)-enantiomer, >99% e.e.], and that fromD. genkwa was 97% e.e. in favor of the (+)-enantiomer. Lignan-synthesizing enzyme activity was detected from a Thymelaeaceae plant for the first time; cell-free extracts fromD. genkwa catalyzed the formation of (–)-lariciresinol (23% e.e.) from racemic (±)-pinoresinols. The stereochemistry of the enzymatic reaction is discussed in relation to the stereochemical features of the isolated lignans.Parts of this report were presented at the 42nd Lignin Symposium, Sapporo, October 1997; 48th Annual Meeting of the Japan Wood Research Society, Shizuoka, April 1998; and 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Isolation and characterization of a cDNA from Catharanthus roseus which is highly homologous with phosphate transporter

The PIT1 gene which is highly homologous with phosphate transporter was isolated from Catharanthus roseus and analyzed. The cBNA PIT1 contained an open reading frame of 542 amino acids and its sequence showed a 31, 30, and 34% identity with the phosphate transporter of Saccharomyces cerevisiae (PBO84), Neurospora crassa (PHO-5), and Glomus versiforme (GvPT), respectively. Furthermore, the cDNA PIT1 encoded a highly hydrophobic protein with 12 putative membrane-spanning regions and contained a conserved amino acid sequence reported in the human glucose transporter super-family* S. cerevisiae strain DpU (pho84 knockout strain) was unable to grow on low phosphate (55 μM) medium (LP medium). Expression of the PIT1 cDNA enabled DpU to grow on LP medium. Northern hybridization analysis revealed that the PIT1 gene was expressed in roots, stems, and young whole plant of C. roseus, but not in leaves.     

Identification of marker genes for intestinal immunomodulating effect of a fructooligosaccharide by DNA microarray analysis

Prebiotic fructooligosaccharides are noted for their intestinal immunodulating effects, and the identification of markers for the effects is a matter of great concern. This study aimed to identify marker genes for physiological effects of a particular fructooligosaccharide (FOS) on a host animal and also to define the target of its function in the small intestine. DNA microarray technology was used to screen candidate marker genes, and comprehensive changes in gene expressions in the ileum of mice fed with FOS were investigated. One of the major physiological effects of FOS was intestinal immunomodulation. Marker genes were then identified for major histocompatibility complex classes I and II, interferon, and phosphatidylinositol metabolites. Also, the ileum was segmented into Peyer's patch (PP) and the other ileal organ (DeltaPP), and these were analyzed by quantitative RT-PCR method, with the result that the site for recognizing the FOS function was the DeltaPP rather than the PP. This is the first paper showing the markers for the physiological effects of FOS in the small intestine at gene expression level. Applying these marker genes would make it possible to clarify the mechanisms of how the administration of dietary FOS and associated changes in the intestinal environment are recognized by host organisms as well as how its immunomodulating effects are expressed in the body.     

Purification and characterization of phytase induced in tomato roots under phosphorus-deficient conditions

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from roots of tomato plants grown under phosphorus-deficient conditions using five purification schemes. The phytase was successfully separated from the major acid phosphatase to an electrophoretic homogeneity. The native molecular weight of this enzyme was estimated to be about 164 kD by Bio-Gel P-200 gel filtration. The molecular weight of the subunit on SDS-PAGE was approximately 82 kD, indicating that the native form of the enzyme was a homodimer. The isoelectric point of tomato phytase was about 5.5. The enzyme exhibited a high affinity for phytic acid (K _m = 38 μM), and was strongly inhibited by phosphate, molybdate and fluoride. Among other characteristics of tomato phytase, the pH and temperature optima were 4.3 and 45°C, respectively. Tomato phytase contained a fairly high concentration of aspartic, glutamic acid and glycine residues.     

Investigation of the short-term effect of chemonucleolysis with chondroitinase ABC.

Chondroitinase ABC (C-ABC) is expected to be a novel agent for chemonucleolysis. The effect of C-ABC was investigated by magnetic resonance (MR) and radiograph. C-ABC was administered into the lumbar intervertebral disks on the clinically normal beagles (n=5), in a dose of 50 microl (12.5 units as C-ABC). MR scans were performed pre-dose, and 1, 3, 7, 14 and 28 days after administration of C-ABC, and the signal intensity (SI) of the nucleus pulposus was measured. Radiographs were taken pre-dose, and 1, 2, 3, 4, 5, 6, 7, 14 and 28 days post-dose, to evaluate narrowing of the disk space in terms of height index (HI). In addition, the quantity of the chondroitin sulfate (CS) and the hyaluronic acid (HA) in the nucleus pulposus were measured by high performance liquid chromatography on day 28 after dosing. SI and HI continuously decreased, following the injection to 37.1% and 78.9% of the pre-dose values, respectively. Statistically significant differences (p<0.01) were observed between the C-ABC group and the control group in the respects on day 1 post-dose. CS and HA contents of the nucleus pulposus were noted to be significantly decreased on day 28 (p<0.01) in the treated group. This agent proved to degenerate proteoglycans in the nucleus pulposus, thus progressively reducing the interdiskal pressure from day 1 post-dose onwards. It is concluded that C-ABC is expected to afford its efficacy from early in the course of chemonucleolysis.