A bitter melon extract inhibits the P-glycoprotein activity in intestinal Caco-2 cells: monoglyceride as an active compound

P-glycoprotein (P-gp) is a 170 kDa membrane protein that belongs to the ATP-binding cassette (ABC) transporter superfamily. In normal tissues, P-gp functions as an ATP-dependent efflux pump that excretes highly hydrophobic xenobiotic compounds, playing an important role in protecting the cells/tissues from xenobiotics. In the present study, chemical substances that could directly modulate the intestinal P-gp activity were searched in vegetables and fruits. By using human intestinal epithelial Caco-2 cells as a model of the small intestinal cells, we observed that a bitter melon fraction extracted from 40% methanol showed the greatest increase of the rhodamine-123 accumulation by Caco-2 cells. Inhibitory compounds in the bitter melon fraction were then isolated by HPLC using Pegasil C4 and Pegasil ODS columns. The HPLC fraction having the highest activity was analyzed by (1)H-NMR and FAB-MS, and the active compound was identified as 1-monopalmitin. It is interesting that certain types of monoglyceride might be involved in the drug bioavailability by specifically inhibiting the efflux mediated by P-gp.     

Establishment of laurel forest vegetation in adjacent secondary forest in central Japan

Distributions of lucidophyllous species are limited due to the fragmentation of laurel forest. On Komayama Hill in central Japan, we evaluated the colonization of typical lucidophyllous vascular plants from a 350-year-old laurel forest into adjacent abandoned secondary forest for conservation and restoration purposes. A total of 14 consecutive subplots were established along the vegetation border between the two forests (length, 30 m; width, 5 m), extending 70 m into the secondary forest; 18 quadrats of old-growth forest were surveyed. Edge effects of old-growth forest were found to play an important role in re-establishing lucidophyllous saplings and seedlings in the secondary forest. In particular, the abundances of the four dominant canopy species of the old-growth forest significantly decreased with increasing distance. Hence, they are expected to colonize further into the secondary forest and, ultimately, to dominate the canopy. However, the number of lucidophyllous species did not change with distance. Species such as Ficus nipponica, Damnacanthus indicus, Ilex integra, and Lemmaphyllum microphyllum were near-completely or completely limited to the old-growth forest. They are known to be negatively affected by forest fragmentation and were observed to be struggling to colonize the exterior of the old-growth forest even after 60 years of abandonment. Their absence highlighted the limited colonization capacities of some old-growth forest species and underlined the time required for habitat restoration following human disturbance. We conclude that it is important to consider the population dynamics of dominant canopy species and the colonization of these interior species when assessing the habitat expansion of lucidophyllous species and hence the restoration of degraded lands.     

Linking two DNA duplexes with a rigid linker for DNA nanotechnology

DNA has recently emerged as a promising material for the construction of nanosized architectures. Chemically modified DNA has been suggested to be an important component of such architectural building blocks. We have designed and synthesized a novel H-shaped DNA oligonucleotide dimer that is cross-linked with a structurally rigid linker composed of phenylene and ethynylene groups. A rotatable DNA unit was constructed through the self-assembly of this H-shaped DNA component and two complementary DNA oligonucleotides. In addition to the rotatable unit, a locked DNA unit containing two H-shaped DNA components was also constructed. As an example of an extended locked structure, a hexagonal DNA origami dimer and oligomer were constructed by using H-shaped DNA as linkers.     

O-Fucose Monosaccharide of Drosophila Notch Has a Temperature-sensitive Function and Cooperates with O-Glucose Glycan in Notch Transport and Notch Signaling Activation

Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1^{R245A knock-in}), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1^{R245A knock-in} and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein''s functions.     

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.     

Isolation of 48 genetic markers appropriate for high throughput genotyping of inbred rat strains by B1 repetitive sequence-representational difference analysis

Mapping of genetic suppressors, modifiers, and quantitative trait loci (QTLs) requires genetic markers that can be efficiently and inexpensively genotyped for a large number of individuals. To isolate rat genetic markers suitable for this purpose, representational difference analysis (RDA) was performed with amplicons prepared by PCR with the B1 repetitive sequence used as the primer (B1-amplicons). In total, 48 polymorphic DNA fragments were isolated by five series of RDA, subtracting the B1-amplicons prepared from an ACI/N (ACI) rat from those prepared from BUF/Nac (BUF), and vice versa. All the polymorphic fragments detected ``presence-or-absence' polymorphisms with B1-amplicons prepared from ACI, BUF, and their F₂ progeny, and each fragment was linkage mapped. Dot-blotting amplicons onto filters at a high density and hybridization of the filters with these B1-RDA markers made it possible to genotype a large number of rats simultaneously for multiple loci. These B1-RDA markers were polymorphic between two given inbred strains of rat at frequencies between 30% and 70%. This is the first report on the isolation of B1-RDA markers among inbred strains of rats. Received: 15 July 1998 / Accepted: 18 August 1998     

Sequence Analysis of Amplified DNA Fragments Containing the Region Encoding the Putative Lipase Substrate-Binding Domain and Genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.