Phylogenetic analyses of Japanese golden chanterelles and a new species description,Cantharellus anzutake sp. nov.

The Japanese golden chanterelle commonly identified as Cantharellus cibarius was sampled in a broad range of forest vegetation. A total of 90 fresh and 11 herbarium specimens were examined microscopically, subjected to sequencing analysis of their nuclear ribosomal RNA (rDNA) and tef-1 genes, and their characteristics were compared with those of European C. cibarius. Based on morphological and ecological characteristics, basidioma samples from Japan were divided into four species. While specimens of Cantharellus sp. 4 from Hokkaido Island were included in the European C. cibarius clade phylogenetically, the other three species formed three unique clades. Among these, Cantharellus anzutake sp. nov. is sister to the clade of C. cibarius and was widely sampled from the northern limit of Honshu Island to the southern limit of Kumejima Island in Ryukyu Islands. Although C. anzutake was morphologically similar to C. cibarius, the two species were phylogenetically distinct. Other morphologically similar but genetically distinct chanterelle species from India exhibited macroscopic and microscopic differences compared with C. anzutake.     

The plant aspartic proteinase-specific polypeptide insert is not directly related to the activity of oryzasin 1.

Many plant aspartic proteinases (APs) are different from animal and microbial APs in that they contain a polypeptide insert, approximately 100 amino acids in length, in the C-terminal region. To interpret the significance of this insert, we constructed an expression system for rice AP oryzasin 1 by linking a pro-oryzasin 1 downstream of glutathione S-transferase (GST). GST-proOS1 expressed the highest degree of hemoglobin-hydrolytic activity when treated at pH 3.3 and incubated for 24 h at room temperature. We carried out a similar experiment using an insert-lacking proOS1 mutant, GST-DeltaproOS1, as the fusion protein, and found it to show similar activity. This result indicates that the insert is not involved in the production of AP activity. We then investigated the autolysis of the two proteins by Western blot analysis. GST-proOS1 was autolyzed into 67- and 64-kDa fragments, while GST-DeltaproOS1 autolyzed to 54- and 52-kDa products. GST-DeltaproOS1 clearly produced two molecular species early in the autolytic process, and not later than 3 h from the start, but no such clear result was observed in the case of GST-proOS1. This suggests that, although the presence of the plant AP-specific insert does not influence the enzyme activity by itself, it apparently has an effect on the autolysis of OS1.     

ADVENTIVE EMBRYOGENESIS IN CITRUS I. THE OCCURRENCE OF ADVENTIVE EMBRYOS WITHOUT POLLINATION OR FERTILIZATION

Anatomical studies of unfertilized undeveloped seeds from open- and control-pollinated fruits of ten facultative apomictic Citrus cultivars were carried out with the aid of light and epifluorescence microscopes. With or without pollination, adventive embryos autonomously developed at all positions in the nucellus in all cultivars. The adventive embryos initiated at the chalazal end of the nucellus were more vigorous than those initiated at the micropylar end. Because of the lack of endosperm and poor seed development, however, all adventive embryos within the unfertilized seeds terminated their development at the globular or early cotyledonary stages and were unable to germinate under natural conditions. The capability of unfertilized seeds to develop varied from species to species. Growth of the adventive embryos was dependent on nucellus size, but the growth rate of adventive embryos relative to nucellus size was different in different species. Neither pollination, fertilization nor subsequent zygote and endosperm development further stimulated adventive embryo initiation. Conversely, pollination and subsequent fertilization of other seeds in the same fruit slightly, but significantly, suppressed adventive embryo growth in the unfertilized seeds. These facts concerning adventive embryogenesis in unfertilized seeds indicate that neither pollination nor fertilization is essential for in vivo adventive embryogenesis and that normal endosperm is necessary for perfect development of adventive embryos initiated only in the micropylar half of the nucellus.     

Expression and localization of aquaporins in rat gastrointestinal tract

A family of water-selective channels, aquaporins (AQP), has beendemonstrated in various organs and tissues. However, the localizationand expression of the AQP family members in the gastrointestinal tracthave not been entirely elucidated. This study aimed to demonstrate theexpression and distribution of several types of the AQP family and tospeculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1-5 and AQP8 was examined in various portions through the gastrointestinal tract.AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon,and their expression was relatively intense in the small intestine andcolon. In contrast, AQP4 mRNA was selectively expressed in the stomachand small intestine and AQP8 mRNA in the jejunum and colon.Immunohistochemistry and in situ hybridization demonstrated cellularlocalization of these AQP in these portions. AQP1 was localized onendothelial cells of lymphatic vessels in the submucosa and laminapropria throughout the gastrointestinal tract. AQP3 was detected on thecircumferential plasma membranes of stratified squamous epithelialcells in the esophagus and basolateral membranes of cardiac glandepithelia in the lower stomach and of surface columnar epithelia in thecolon. However, AQP3 was not apparently detected in the smallintestine. AQP4 was present on the basolateral membrane of the parietalcells in the lower stomach and selectively in the basolateral membranesof deep intestinal gland cells in the small intestine. AQP8 mRNAexpression was demonstrated in the absorptive columnar epithelial cellsof the jejunum and colon by in situ hybridization. These findings mayindicate that water crosses the epithelial layer through these waterchannels, suggesting a possible role of the transcellular route forwater intake or outlet in the gastrointestinal tract.     

Identification of Stmm3 locus Conferring Resistance to Late-stage Chemically Induced Skin Papillomas on Mouse Chromosome 4 by Congenic Mappingand Allele-specific Alteration Analysis

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F₁ × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N₂ and N₁₀ congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development.     

Oxidation of low-density lipoprotein by copper and iron in phosphate buffer

We examined the effect of phosphate buffer on the iron- and copper-catalyzed peroxidation of low-density lipoprotein (LDL). The incubation of LDL with CuSO4 in 0.15 M NaCl led to the peroxidation of LDL as evidenced by the detection of thiobarbituric acid-reactive substances (TBARS) and lipid hydroperoxides (LPO). The peroxidation of LDL was also observed with FeSO4 and FeCl3 in 0.15 M NaCl, although there was a lag phase with FeCl3. In 10 mM phosphate buffer, the peroxidation of LDL was observed with CuSO4 to an extent similar to that in 0.15 M NaCl. However, the peroxidation induced by incubation with FeSO4 and FeCl3 was significantly inhibited in phosphate buffer. Iron and copper each formed a complex with lipoprotein during incubation with LDL in 0.15 M NaCl. Although no effect on the formation of copper-LDL complex was observed in phosphate buffer, the formation of iron-LDL complex was reduced in the buffer. These observations suggest there are marked differences in the peroxidation of LDL and in the formation of complexes with LDL between iron and copper in phosphate buffer.