Oncostatin M stimulates proliferation and functions of mouse fetal liver cells in three-dimensional cultures

In order to develop a tissue engineered bioartificial liver (BAL), long-term three-dimensional (3-D) culture of fetal liver cells (FLCs) utilizing porous polymer as a scaffold was performed for up to 1 month. The effects of the basal medium and supplementation with oncostatin M (OSM) on the proliferation and differentiation of mouse FLCs were examined in both 3-D culture and conventional monolayer dish culture. Compared with monolayer culture, cell numbers and hepatic function of FLCs were better maintained by 3-D culture. When two kinds of basal media were tested in this study, Williams' medium E (WE) was superior to minimum essential medium alpha (alphaMEM) in expressing hepatic function of FLCs in both 3-D and monolayer cultures, although higher cell densities were obtained with alphaMEM. OSM potently stimulated both cell growth and metabolic activity, especially in 3-D culture. When WE supplemented with OSM was used for 3-D culture, albumin secretion by FLCs increased dramatically after day 5, and a high level of secretion was maintained until the end of culture. During a period of over 1 month, no decrease of albumin secretion was observed. In conclusion, this 3-D culture method was expected to be one of the realistic attempts to develop a tissue engineered BAL.     

Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes

Cationic amphiphilic drugs (CADs) cause massive intracellular accumulation of phospholipids, thereby resulting in phospholipidosis (PLD); however, the molecular mechanism underlying CAD-induced PLD remains to be resolved. Here, we found that treatment of normal rat kidney cells with CADs known to induce PLD caused redistribution of a mannose 6-phosphate/IGF-II receptor (MPR300) from the TGN to endosomes and concomitantly increased the secretion of lysosomal enzymes, resulting in a decline of intracellular lysosomal enzyme levels. These results enable the interpretation of why CADs cause excessive accumulation of undegraded substrates, including phospholipids in lysosomes, and led to the conclusion that the impaired MPR300-mediated sorting system of lysosomal enzymes reflects the general mechanism of CAD-induced PLD. In addition, our findings suggest that the measurement of lysosomal enzyme activity secreted into culture medium is useful as a rapid and convenient in vitro early screening system to predict drugs that can induce PLD.     

Separation and Characterization of Thylakoid and Cell Envelope of the Blue-green Alga (Cyanobacterium) Anacystis nidulans

The thylakoid and the cell envelope of the blue-green alga Anacystisnidulans were separated by mechanical disruption of lysozyme-treatedcells followed by differential and density gradient centrifugation.The prepared envelope was composed of an outer membrane, a peptidoglycanlayer and possibly a part of the cytoplasmic membrane. The preparedthylakoid retained the size and intricate structure typicalof the thylakoid membrane of this alga. Light absorption andfluorescence spectra revealed that the envelope contained carotenoids,a pigment with an absorption maximum at 748 nm (P750), and asmall amount of pheophytin-like pigment with an absorption maximumat 673 nm. The thylakoid contained chlorophyll a and carotenoidsbut no P750. The thylakoid contained five kinds of carotenoids,the major ones being rß-carotene and zeaxanthin, whereasthe cell envelope contained two kinds of carotenoids, zeaxanthinand nostoxanthin. Four kinds of lipids, abundant in the blue-greenalgae, were present in both the thylakoid and the cell envelope.However, the content of sulfolipid was very low in the cellenvelope. The polypeptide compositions differed between thethylakoid and the cell envelope. Similarities between blue-greenalgal cells and eukaryotic chloroplasts are discussed with respectto the spectrophotometric and biochemical characteristics ofthe thylakoid and the envelope. (Received March 7, 1981; Accepted May 22, 1981)     

ABO Blood Type and Personality Traits in Healthy Japanese Subjects

There is no scientific consensus that a relationship exists between the ABO blood group and personality traits. However, a recent study hypothesized that the dopamine beta-hydroxylase (DBH) gene is in linkage with the ABO gene. The sample population consisted of 1,427 healthy Japanese subjects who completed the Temperament and Character Inventory (TCI). Each subject’s ABO blood type was determined by genotyping the rs8176719 and rs8176746 ABO gene single-nucleotide polymorphisms (SNPs) using a TaqMan genotyping assay. The relationships between the six ABO genotypes or four ABO phenotypes and personality traits were examined using a multivariate analysis of covariance (MANCOVA), controlling for age and sex. The MANCOVA data showed a significant difference in TCI scores among the ABO genotype groups (F [7, 1393] = 3.354, p = 0.001). A subsequent univariate analysis showed a significant difference in the mean scores for Persistence among the genotype groups (F = 2.680, partial η² = 0.010, p = 0.020). Similarly, dividing the ABO blood type into four phenotypes revealed a significant difference among the phenotype groups (F [7, 1397] = 2.529, p = 0.014). A subsequent univariate analysis showed a significant difference among the phenotype groups in the mean scores for Persistence (F = 2.952, partial η²= 0.006, p = 0.032). We observed a significant association between ABO blood group genotypes and personality traits in a large number of healthy Japanese subjects. However, these results should be regarded as preliminary and should be interpreted with caution because it is possible that the association between ABO blood group genotype and the Persistence trait is relatively weak.     

Impact of N-Saturated Upland Forests on Downstream N Pollution in the Tatara River Basin, Japan

This study evaluated whether nitrogen (N) saturated upland forests can degrade downstream water quality in the Tatara River Basin, northern Kyushu, western Japan. Our hypothesis is that elevated atmospheric N deposition degrades downstream water quality in a watershed containing N-saturated forests because a considerable amount of atmospherically deposited N passes into the streams without being retained. Synoptic stream water samplings were conducted at 23 sites across a wide range of land-use categories in the basin over 1 year. A long-term temporal analysis of downstream water quality over the last 30 years (1977–2007) was conducted and compared with long-term trends in related factors such as urban/agricultural activity, sewage wastewater treatment, atmospheric N deposition, and forest condition. The results showed that atmospherically deposited N to N-saturated forests can be a large enough non-point source of N leaving the watershed to impact downstream water quality. This was highlighted by the reduction in pollutant exports derived from urban/agricultural activities, an increase in atmospheric N deposition, and the maturation of coniferous plantation forests in the past 30 years. These have led to reductions in total phosphorus and organic nitrogen concentrations in downstream water, whereas downstream nitrate (NO₃ ⁻) concentrations increased over the last 30 years. The consequent increase in the downstream N:P ratio indicated P limitation. Reducing the NO₃ ⁻ exports from N-saturated upland forests is suggested as a strategy to improve regional downstream NO₃ ⁻ pollution, but involves intercontinental-scale action in reducing atmospheric N emissions.     

GROWTH AND DIFFERENTIATION OF MITOCHONDRIA IN THE REGENERATING RAT ADRENAL CORTEX : A Correlated Biochemical and Stereological Approach

Diameters of the circular profiles of spherical mitochondria in parenchymal cells of the zona fasciculata in rat adrenal cortex were measured for intact controls and for the regenerating adrenal cortex on electron micrographs recorded at random. The diameter data were then processed by Bach's method which deals with the sphere size distribution. The structural parameters of the mitochondria were computed with the aid of an electronic computer. The total number of mitochondria in all the parenchymal cells of the zona fasciculata were calculated. The surface area of the inner mitochondrial membrane was then determined stereologically. Biochemical parameters were obtained for the protein, the phospholipid, and the cytochrome P-450 content, per averaged mitochondrion. The number of cytochrome P-450 molecules contained in the inner membrane was determined in terms of the unit surface area and of the unit amount of phospholipid. These correlated biochemical and stereological parameters have led to the following conclusions. (a) The genesis of the mitochondria after the adrenal enucleation is almost completed within 10 days. (b) During the period of mitochondrial proliferation, the mitochondria are small in size and also immature both in the structure and in the function of their inner membrane, (c) These small and immature mitochondria grow through an increase of the phospholipid and protein, and this increase is accompanied by expansion of the area of the membrane surface, (d) An enrichment of the inner membrane with cytochrome P-450 molecules occurs, thus indicating the differentiation of adrenocortical mitochondria. The process of membrane differentiation is not tightly coupled with that of membrane growth.     

Functional analysis of the carbohydrate recognition domains and a linker peptide of galectin-9 as to eosinophil chemoattractant activity

Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.     

Characterization of the sperm-associated body and its role in the fertilization of the chicken Gallus domesticus

The present paper aimed to characterize the substance forming the sperm-associated body (SB), to find its producing sites, and to show its functions in the fertilization of chicken. The SB was found both in between the inner and outer layers of vitelline membranes around eggs and in the oviductal infundibulum. Material from which the SB is constructed (SB substance) was isolated from the vitelline membranes. It was a hydrophobic protein with a molecular size of 570 kDa. X-ray microanalysis detected calcium in the aggregates of the SB substance. Immunofluorescence and immunoelectron microscopy showed that the substance was produced in secretory cells in the luminal epithelium of the oviductal infundibulum and was provided to the egg on and in its vitelline membrane. During incubation, the SB substance bound with spermatozoa in the posterior portion of their flagella. Holes and disks were found in the vitelline membranes of fertile eggs at a ratio of 1: 19-24. Over 94% of the holes were accompanied by SB. The presence of SB is necessary for fertile spermatozoa to make holes in the membrane and to enter the fertile egg.     

Gene amplification profiling of esophageal squamous cell carcinomas by DNA array CGH

Gene amplification is one of the basic mechanisms that lead to overexpression of oncogenes. DNA array comparative genomic hybridization (CGH) has great potential for comprehensive analysis of both a relative gene-copy number and altered chromosomal regions in cancers, which enables us to identify new amplified genes and unstable chromosomal loci. We examined the amplification status in 32 esophageal squamous cell carcinomas (ESCCs) and 13 ESCC cell lines on 51 frequently amplified loci in a variety of cancers by both DNA array CGH and Southern blot analyses. The 1p34 locus containing MYCL1, 2p24 (MYCN), 7p12 (EGFR), and 12q14 (MDM2) were amplified in one of the 32 cases (3%), and the 17q12 locus (ERBB2) and 8p11 (FGFR1) in two of the 32 cases (6%), while only the 11q13 locus (Cyclin D1, FGF4, and EMS1) was frequently amplified (28%, 9/32), demonstrating this locus to be a major target in ESCCs. One locus, 8q24 (c-MYC) was found to be amplified only in the cell lines. Eight out of 51 loci (15.7%) were found to be amplified in at least one of the 32 primary ESCCs or the 13 ESCC cell lines, suggesting that chromosomal loci frequently amplified in a type of human cancer may also be amplified in other types of cancers. This paper is the first report of an application of DNA array CGH to ESCCs.