Effects of sphingosine 2N- and 3O-methylation on palmitoyl ceramide properties in bilayer membranes

To study the role of the interfacial properties of ceramides in their interlipid interactions, we synthesized palmitoylceramide (PCer) analogs in which a methyl group was introduced to the amide-nitrogen or the C3-oxygen of the sphingosine backbone. A differential scanning calorimetry analysis of equimolar mixtures of palmitoylsphingomyelin (PSM) and PCer showed that these sphingolipids formed a complex gel phase that melted between 67°C and 74°C. The PCer analogs also formed gel phases with PSM, but they melted at lower temperatures compared with the system with PCer. In complex bilayers composed of an unsaturated glycerophospholipid, PSM, and cholesterol, the 3O-methylated ceramide formed a cholesterol-poor ordered phase with PSM. However, the 2N-methylated and doubly methylated (2N and 3O) PCer analogs failed to displace sterol from interactions with PSM. Like PCer, the analogs reduced sterol affinity for the complex bilayers, but this effect was most pronounced for the 3O-methylated ceramide. Taken together, our results show that 2N-methylation weakened the ceramide-PSM interactions, whereas the 3O-methylated ceramide behaved more like PCer in interactions with PSM. Our findings are compatible with the view that interlipid interactions between the amide-nitrogen and neighboring lipids are important for the cohesive properties of sphingolipids in membranes, and this also appears to be a valid model for ceramide.     

Acetylation regulates monopolar attachment at multiple levels during meiosis I in fission yeast

In fission yeast, meiotic mono-orientation of sister kinetochores is established by cohesion at the core centromere, which is established by a meiotic cohesin complex and the kinetochore protein Moa1. The cohesin subunit Psm3 is acetylated by Eso1 and deacetylated by Clr6. We show that in meiosis, Eso1 is required for establishing core centromere cohesion during S phase, whereas Moa1 is required for maintaining this cohesion after S phase. The clr6-1 mutation suppresses the mono-orientation defect of moa1Δ cells, although the Clr6 target for this suppression is not Psm3. Thus, several acetylations are crucial for establishing and maintaining core centromere cohesion.     

Electrical properties of dendritic spines with bulbous end terminals

Several suggestions have been made about the functional significance of dendritic spines in connection with synaptic plasticity. We investigated transient electrical behavior of spines with bulbous terminals in neurons with arbitrary dendritic geometries. It is shown that postsynaptic potential transform caused by a synapse on a spine can be resolved into a product of two transfer functions and the synaptic input current transform. The first transfer function was determined to be independent of the spine. The second transfer function represents the straightforward attenuation effect of the spine, which determines the effective synaptic current reaching the parent dendrite. Using what is known of the size and the shape of spines from histology, we conclude that almost all of the synaptic current flow into the parent dendrite, and that therefore the straightforward attenuation effect is negligible. Consequently, when the synaptic current remained unaltered, as was the case for a large synaptic resistance as compared with the spine stem resistance, a morphological change of the spine did not produce an effective change in the postsynaptic potential. On the other hand, when the synaptic resistance is compared with the spine stem impedance, the morphological change of the spine might induce changes of the synaptic current and the postsynaptic potential.     

Pyruvate:NADP+ oxidoreductase is stabilized by its cofactor,thiamin pyrophosphate,in mitochondria of Euglena gracilis

Pyruvate:NADP(+) oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.     

Improved culture conditions for somatic embryogenesis from Asparagus officinalis L. using an aseptic ventilative filter

Summary Calli were induced from the crown of seedlings or lateral bud of young spears of Asparagus officinalis L. on Linsmaier and Skoog's (LS) solid-medium supplemented with 5 M 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic callus was selected from induced calli and proliferated in LS liquid medium supplemented with 5 M 2,4-D. Non-vitrified somatic embryos were formed and efficiently developed into club-shaped embryos in LS hormone-free medium with 1 % gelrite in a culture vessel capped with an aseptic ventilative filter. Non-vitrified club-shaped embryos developed into normal plants when transferred to half-strength LS medium without hormones, and 0.8 % agar. Carbon dioxide concentration and moisture content inside the culture vessels were measured, and their effect on embryo development is discussed.     

Molecular and enzymatic properties of 1,2-α-d-mannosidase fromAspergillus saitoi

The molecular properties, such as molecular weight, N-and C-terminal amino acids, amino acid composition, and circular dichroism, of 1,2--mannosidase isolated from the culture filtrate ofAspergillus saitoi were determined.The enzyme had aK _m of 0.67 mM andk _cat of 1.27/s with mannobiose at pH 50.0 and 30°C. The anomeric configuration of the reaction products of the enzyme was examined by studying the -anomer. A single Manl2Man linkage in intact Taka-amylase A fromAspergillus oryzae was hydrolyzed, producing free mannose.