Electron microscopic study of subcapsular cell hyperplasia in the adrenal glands of IQI/Jic mice.

Hyperplastic cells in subcapsular cell hyperplasia (SCH) lesion in adrenal glands of female IQI/Jic mice were examined by electron microscopy. These cells were small and polygonal, and had irregular nuclei, elongated mitochondria with lamellar cristae and dense lipid droplets. While these cells showed different features, some of them had desmosomes and basement membranes, and a few round mitochondria with tubular cristae as endocrine cells. These findings suggest that hyperplastic cells in SCH lesions might originate from endocrine blastemic cells.     

Liver gangliosides of various animals ranging from fish to mammalian species

Liver gangliosides of different animal species were analyzed. Bony fish liver contained a major ganglioside that migrated faster than GM3 on thin-layer chromatography (TLC). This ganglioside was identified to be GM4 (NeuAc) by methods including product analysis after sialidase treatment and negative-ion electrospray ionization (ESI)-mass spectrometry (MS). The presence of GM4 (NeuGc) in fish liver was also demonstrated. The main ganglioside band of bovine liver consisted of two different molecular species, i.e. GD1a (NeuAc/NeuAc) and GD1a (NeuAc/NeuGc). Major gangliosides of liver tissue exhibited a distinct phylogenetic profile; GM4 was expressed mainly in lower animals such as bony fish and frog liver, whereas mammalian liver showed ganglioside patterns with smaller proportions of monosialo ganglioside species. While c-series gangliosides were consistently expressed in lower animals, they were found only in mammalian liver of particular species. No apparent trend was observed between the concentration of liver gangliosides and the phylogenetic stage of animals. The present study demonstrates the species-specific expression of liver gangliosides.     

Molecular Characterization of Lactobacillus plantarum Genes for β-Ketoacyl-Acyl Carrier Protein Synthase III (fabH) and Acetyl Coenzyme A Carboxylase (accBCDA), Which Are Essential for Fatty Acid Biosynthesis

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded β-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the β and α subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.     

A pollen-expressed gene for a novel protein with an F-box motif that is very tightly linked to a gene for S-RNase in two species of cherry,Prunus cerasus and P. avium

This study describes a novel F-box protein gene in the S-locus of sour cherry (Prunus cerasus) and sweet cherry (P. avium). The gene showed an S-haplotype-specific sequence polymorphism and the expression was specific to pollen. Genomic DNA blot analysis of eight sweet cherry cultivars with the probe for the F-box protein gene under low stringency conditions yielded RFLP bands specific to the S-haplotypes of each cultivar. We discuss the possibility of the gene for the F-box protein being a candidate for the male determinant of gametophytic self-incompatibility in PRUNUS:     

Occurrence and tissue distribution of c-series gangliosides in the common squid Todarodes pacificus

We have recently demonstrated that the common squid Todarodes pacificus express acidic lipids that were reactive with a monoclonal antibody A2B5. In the present study, two A2B5-reactive acidic lipids were isolated from squid hepatopancreatic tissue and characterized for their structures by methods including glycolipid overlay analysis, product analysis after sialidase treatment, and electrospray ionization-mass spectrometry (ESI-MS). Accordingly, the two acidic lipid were identified as GT3 and GQ1c, respectively. Another A2B5-reactive acidic lipid in the tissue was tentatively assigned to GT2 based upon its reactivity to A2B5 and chromatographic mobility on thin-layer chromatography. The composition and concentration of c-series gangliosides significantly differed among squid tissues (i.e. hepatopancreas, cerebral ganglion, eye lens, and mantle tissue). Interestingly, the percentages of c-series gangliosides within total gangliosides of hepatopancreas and cerebral ganglion were even higher than that of cod fish brain, which is known to be highly enriched with this ganglioside species. These findings strongly support the hypothesis that c-series gangliosides in squid tissues are not derived from ganglioside-containing food intake, but biosynthesized in a tissue-specific manner.     

The use of the S haplotype-specific F-box protein gene,SFB, as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot (Prunus mume)

Japanese apricot (Prunus mume) exhibits the S-RNase-based gametophytic self-incompatibility system as do other self-incompatible Prunus species. This report identifies the S haplotype-specific F-box protein gene (SFB), a candidate gene for pollen-S, of Japanese apricot, which leads to the development of a molecular typing system for S-haplotype in this fruit species. Both 5- and 3-RACE (rapid amplification of cDNA ends) were performed with SFB gene-specific oligonucleotide primers to clone Pm-SFB¹ and Pm-SFB⁷ of 'Nanko (S¹S⁷)'. As in the case of SFB of other Prunus species, Pm-SFB¹ and Pm-SFB⁷ showed a high level of S-haplotype-specific sequence polymorphism and their expression was specific to pollen. Genomic DNA-blot analyses of 11 Japanese apricot cultivars with the Pm-SFB probes under low stringency conditions yielded RFLP bands specific to the S¹- to S⁸-haplotypes as well as a self-compatible S^f-haplotype. A practical usage of SFB as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot is discussed.Communicated by H.F. LinskensThe nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank/DDBJ database under accession numbers, AB101440 and AB101441, for SFB¹ and SFB⁷, respectively     

Optimal combination of soluble factors for tissue engineering of permanent cartilage from cultured human chondrocytes

Since permanent cartilage has poor self-regenerative capacity, its regeneration from autologous human chondrocytes using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders. However, the conventional autologous chondrocyte implantation is insufficient both in quantity and in quality due to two major limitations: dedifferentiation during a long term culture for multiplication and hypertrophic differentiation by stimulation for the redifferentiation. To overcome the limitations, this study attempted to determine the optimal combination in primary human chondrocyte cultures under a serum-free condition, from among 12 putative chondrocyte regulators. From the exhaustive 2(12) = 4,096 combinations, 256 were selected by fractional factorial design, and bone morphogenetic protein-2 and insulin (BI) were statistically determined to be the most effective combination causing redifferentiation of the dedifferentiated cells after repeated passaging. We further found that the addition of triiodothyronine (T3) prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes via the suppression of Akt signaling. The implant formed by the human chondrocytes cultured in atelocollagen and poly(l-latic acid) scaffold under the BI + T3 stimulation consisted of sufficient hyaline cartilage with mechanical properties comparable with native cartilage after transplantation in nude mice, indicating that BI + T3 is the optimal combination to regenerate a clinically practical permanent cartilage from autologous chondrocytes.     

Self-compatible peach (<Emphasis Type="Italic">Prunus persica</Emphasis>) has mutant versions of the <Emphasis Type="Italic">S</Emphasis> haplotypes found in self-incompatible <Emphasis Type="Italic">Prunus</Emphasis> species

This study demonstrates that self-compatible (SC) peach has mutant versions of S haplotypes that are present in self-incompatible (SI) Prunus species. All three peach S haplotypes, S ¹ , S ² , and S ^2m , found in this study encode mutated pollen determinants, SFB, while only S ^2m has a mutation that affects the function of the pistil determinant S-RNase. A cysteine residue in the C5 domain of the S ^2m -RNase is substituted by a tyrosine residue, thereby reducing RNase stability. The peach SFB mutations are similar to the SFB mutations found in SC haplotypes of sweet cherry (P. avium) and Japanese apricot (P. mume). SFB ¹ of the S ¹ haplotype, a mutant version of almond (P. dulcis) S ^k haplotype, encodes truncated SFB due to a 155 bp insertion. SFB ² of the S ² and S ^2m haplotypes, both of which are mutant versions of the S ^a haplotype in Japanese plum (P. salicina), encodes a truncated SFB due to a 5 bp insertion. Thus, regardless of the functionality of the pistil determinant, all three peach S haplotypes are SC haplotypes. Our finding that peach has mutant versions of S haplotypes that function in almond and Japanese plum, which are phylogenetically close and remote species, respectively, to peach in the subfamily Prunoideae of the Roasaceae, provides insight into the SC/SI evolution in Prunus. We discuss the significance of SC pollen part mutation in peach with special reference to possible differences in the SI mechanisms between Prunus and Solanaceae.     

Extraction and preliminary characterization of maturation-promoting factor from starfish oocytes

Cytoplasm of maturing starfish oocytes possesses a factor which induces maturation upon injection into immature oocytes. Such maturation-promoting factor (MPF) was extracted from maturing oocytes of Asterina pectinifera and characterized preliminarily. After 1-methyladenine (1-MeAde) treatment, maturing oocytes were packed in a centrifuge tube to remove jelly and excess medium, and then crushed by centrifugation. The turbid supernatant was homogenized with a buffer containing NaF, Na-beta-glycerophosphate, ATP, EGTA and leupeptin, followed by centrifugation. MPF extracted in the supernatant was purified partially by ammonium sulfate precipitation, hydrophobic chromatography on pentyl-agarose and gel filtration on Sephacryl S-300. The final material induced maturation in the recipient starfish oocytes when 0.5 ng of protein was injected in a volume of 400 pl. The maturation response included germinal vesicle breakdown, and formation of polar bodies and egg pronucleus. Such MPF preparation induced maturation in oocytes of Xenopus laevis as well. Further, starfish MPF was found to be a heat-labile protein; its molecular weight (MW) was estimated as 300 X 10(3) D by gel filtration and its sedimentation coefficient value as 5S by centrifugation on sucrose density gradients.