Galactosylceramide- and lactosylceramide-loading studies in cultured fibroblasts from normal individuals and patients with globoid cell leukodystrophy (Krabbe's disease) and GM1-gangliosidosis

The metabolism of galactosylceramide and lactosylceramide in cultured fibroblasts was studied using the lipid-loading test. These compounds were incorporated into the fibroblasts yet only small amounts of the incorporated lipids were hydrolyzed unless additional phospholipid was mixed with the glycolipid before loading. Among phospholipids, phosphatidylserine was the most effective for incorporation and hydrolysis of the glycolipids, while phosphatidylcholine inhibited the incorporation of the glycolipids. Using filtration techniques, light scattering analyses and subcellular fractionation, the particle size of glycolipid in the culture medium was found to be critically important for the incorporation of the lipids into the cells and their transportation to the lysosomes. The particle sizes of the glycolipids were decreased by mixing with phosphatidylserine. Furthermore, the negative charge in phosphatidylserine may be necessary for the glycolipid transportation into the lysosomes. In fibroblasts from patients with globoid cell leukodystrophy, 40-50% of galactosylceramide was hydrolyzed on the 4th day of culture, a time when the control fibroblasts had hydrolyzed it about 80%. This finding is in contrast with observations made on fibroblasts with other sphingolipidoses which showed near-zero degradation in corresponding substrate-loading tests. In fibroblasts from patients with either globoid cell leukodystrophy of GM1-gangliosidosis, hydrolysis of lactosylceramide was fairly normal yet somewhat lower than control values on any day of culture, thereby indicating that, in the loading tests, lactosylceramide seems to be hydrolyzed with similar levels of enzyme activities by two distinct beta-galactosidases, galactosylceramidase and GM1-ganglioside beta-galactosidase.     

Cyclic loading on the red cell membrane in a shear flow: a possible cause of hemolysis

The fluid force acting on single human red cells in a high shear flow was analyzed. A two-dimensional elliptical microcapsule as a model of the deformed red cells was adopted to numerically calculate the distributions of the shear forces on both sides of the cell membrane. It is theoretically shown that the cell membrane undergoes an unsteady cyclic loading under the rotational motion around the interior. The mechanism leading to blood cell trauma is examined by repeatedly loading the continuously moving cell membrane.     

Association analysis of salt tolerance in cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp) at germination and seedling stages

Key message

This is the first report on association analysis of salt tolerance and identification of SNP markers associated with salt tolerance in cowpea.

Abstract

Cowpea (Vigna unguiculata (L.) Walp) is one of the most important cultivated legumes in Africa. The worldwide annual production in cowpea dry seed is 5.4 million metric tons. However, cowpea is unfavorably affected by salinity stress at germination and seedling stages, which is exacerbated by the effects of climate change. The lack of knowledge on the genetic underlying salt tolerance in cowpea limits the establishment of a breeding strategy for developing salt-tolerant cowpea cultivars. The objectives of this study were to conduct association mapping for salt tolerance at germination and seedling stages and to identify SNP markers associated with salt tolerance in cowpea. We analyzed the salt tolerance index of 116 and 155 cowpea accessions at germination and seedling stages, respectively. A total of 1049 SNPs postulated from genotyping-by-sequencing were used for association analysis. Population structure was inferred using Structure 2.3.4; K optimal was determined using Structure Harvester. TASSEL 5, GAPIT, and FarmCPU involving three models such as single marker regression, general linear model, and mixed linear model were used for the association study. Substantial variation in salt tolerance index for germination rate, plant height reduction, fresh and dry shoot biomass reduction, foliar leaf injury, and inhibition of the first trifoliate leaf was observed. The cowpea accessions were structured into two subpopulations. Three SNPs, Scaffold87490_622, Scaffold87490_630, and C35017374_128 were highly associated with salt tolerance at germination stage. Seven SNPs, Scaffold93827_270, Scaffold68489_600, Scaffold87490_633, Scaffold87490_640, Scaffold82042_3387, C35069468_1916, and Scaffold93942_1089 were found to be associated with salt tolerance at seedling stage. The SNP markers were consistent across the three models and could be used as a tool to select salt-tolerant lines for breeding improved cowpea tolerance to salinity.

     

Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system.

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure -->2-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1-->3)-alpha- Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfbK and rfbM, were identified. They correspond to six open reading frames (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 amino acids. They are all transcribed in the counter direction to those of the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coli strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitro synthesis of the O9 mannan in membranes isolated from the mutants, showed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannosyl transferases. MtfC (from ORF274), the first mannosyl transferase, transfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenous acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the number of sugars in the repeating unit. Analysis of the predicted amino acid sequence of the ORF261 and ORF431 proteins indicated that they function as components of an ATP-binding cassette transport system. A possible correlation between the mechanism of polymerization and mode of membrane translocation of the products is discussed.     

Studies on cyclic peptides. II. Synthesis of cyclic peptides containing sarcosine.

Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)₂, cyclo-(Sar-Sar-Gly)₂, cyclo-(Sar₄), and cyclo-(Sar₆) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the N^α-protecting group, which was removed by acid. Benzyl ester was used to protect the C-terminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. Deblocking and cyclization of the linear peptides gave the cyclic peptides.     

Amino acid sequence of thermostable direct hemolysin produced by Vibrio parahaemolyticus

The complete amino acid sequence of the subunit of thermostable direct hemolysin, a dimeric protein composed of identical subunits isolated from Vibrio parahaemolyticus, was determined by sequencing BrCN-peptides, their tryptic peptides, and overlaps obtained by Achromobacter protease I digestion. The subunit consists of 165 amino acid residues with the sole disulfide bond between Cys 151 and Cys 161. It is deduced that the biologically active hemolysin is formed by noncovalent association of subunits which are not linked together by disulfide bonds. The primary structure of hemolysin elucidated in the present study is essentially the same as that deduced from the nucleotide sequence of a gene encoding the protein but differs in 9 amino acid residues, suggesting the possibility of the presence of multiple genes for the thermostable direct hemolysin in Vibrio parahaemolyticus.     

Proliferation of unilocular fat cells in the primary culture

Mature white fat cells (unilocular fat cells) have generally been considered to be in terminal differentiation and, hence, to have no proliferative ability. A new method, referred to as "ceiling culture," has been devised in our laboratory to culture unilocular fat cells in vitro. Under such culture conditions, the fat cells continue to exhibit specific functions of lipid metabolism and proliferate extensively. Intracytoplasmic lipid droplets did not inhibit division of the cells. There were two modes of proliferation of unilocular fat cells: "loculus-dividing" cell division, in which the single loculus of fat in the dividing cell was broken down into multiple droplets and distributed evenly between the daughter cells, and "loculus-preserving" cell division, in which the loculus in the dividing cell was minimally broken down and inherited with its shape preserved by one of the daughter cells with the other getting only a small number of fine lipid droplets. Such findings suggest that unilocular fat cells in mature fat tissue in vivo are probably capable of proliferation in such modes under some conditions.     

Seed dispersal by pygmy chimpanzees (Pan paniscus): A preliminary report

The role in seed dispersal played by the pygmy chimpanzees (Pan paniscus) inhabiting Wamba, Republic of Zaïre, was studied. Germination was tested for seeds of 17 plant species recovered from the feces of pygmy chimpanzees at Wamba. The fecal seeds of 13 species germinated, and in six of the species the germination rate for the fecal seeds was higher than that of control seeds. Although five other species showed a higher germination rate in the control seeds than in the fecal seeds, the remaining two species revealed no difference in germination rate between the fecal and control seeds. There was no great difference in germination velocity between the fecal and control seeds of the same species. For comparison, seeds of four plant species collected from the feces of common chimpanzees (Pan troglodytes) and gibbons (Hylobates lar) in captivity in Okinawa were tested for their germinability. In this test, although the seeds had passed through the digestive tract, their germinability demonstrated little change. Based on the behavioral characteristics of the pygmy chimpanzee at Wamba and observations of the captive primates on Okinawa, it seems that pygmy chimpanzees may play an important role in the seed dispersal of fruit plant species at Wamba.