Accumulation of neutral lipids in Saccharomyces carlsbergensis by myo-inositol deficiency and its mechanism. Reciprocal regulation of yeast acetyl-CoA carboxylase by fructose bisphosphate and citrate.

The abnormal accumulation of lipids due to myo-inositol deficiency in Saccharomyces carlsbergensis, and the mechanism involved was investigated. The deficient cells contained much more neutral lipids with a greater ratio of unsaturated fatty acids compared to the supplemented cells, whereas there was no significant change in their phospholipid contents. The biosynthesis of fatty acids and sterols from acetate, and of triacylglycerols and sterol esters from palmitate was markedly augmented in the deficient cells. Acetyl-CoA carboxylase activity of the deficient supernatant was 2- to 5-fold higher than that of the supplemented. However, the activity from both sources was not significantly different after Sephadex G-25 gel filtration of the supernatant, suggesting the presence of low molecular effector(s) in the deficient supernatant. There was a great increase in acid-soluble glycogen, trehalose, and fructose-1,6-P2, as well as a drastic decrease in citrate in the deficient cells. Their intracellular levels were calculated so that their effects on acetyl-CoA carboxylase was examined over the range of physiological concentration. Citrate strongly inhibited the enzyme activity of the supernatant, but it had no effect on the preparation after gel filtration. On the other hand, fructose-1,6-P2 stimulated the enzyme activity both before and after gel filtration. The acetyl-CoA carboxylase activity in the gel filtrate was measured as a function of citrate concentration at several fixed concentrations of fructose-1,6-P2. Citrate counteracted the activation by fructose-1,6-P2 in a dose-dependent manner. Citrate lacked the inhibitory effect in the absence of fructose-1,6-P2. It was concluded from these results that neutral lipid accumulation in the deficient cells reflected an increase in the synthesis of fatty acids, at least partly based on an enhancement of acetyl-CoA carboxylase activity, and that the operation of a reciprocal regulation of the enzyme by fructose-1,6-P2 and citrate caused a marked elevation of the enzyme activity in the deficient cells with a high fructose-1,6-P2 level and a low citrate level.     

Critical requirement for the membrane-proximal cytosolic tyrosine residue for CD28-mediated costimulation in vivo

The YMNM motif that exists in the CD28 cytoplasmic domain is known as a binding site for phosphatidylinositol 3-kinase and Grb-2 and is considered to be important for CD28-mediated costimulation. To address the role of the YMNM motif in CD28 cosignaling in primary T cells, we generated transgenic mice on a CD28 null background that express a CD28 mutant lacking binding ability to phosphatidylinositol 3-kinase and Grb-2. After anti-CD3 and anti-CD28 Ab stimulation in vitro, the initial proliferative response and IL-2 secretion in CD28 Y189F transgenic T cells were severely compromised, while later responses were intact. In contrast to anti-CD3 and anti-CD28 Ab stimulation, PMA and anti-CD28 Ab stimulation failed to induce IL-2 production from CD28 Y189F transgenic T cells at any time point. Using the graft-vs-host reaction system, we assessed the role of the YMNM motif for CD28-mediated costimulation in vivo and found that CD28 Y189F transgenic spleen cells failed to engraft and could not induce acute graft-vs-host reaction. Together, these results suggest that the membrane-proximal tyrosine of CD28 is required for costimulation in vivo. Furthermore, these results indicate that the results from in vitro assays of CD28-mediated costimulation may not always correlate with T cell activation in vivo.     

Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons

AMPA-type glutamate receptors (AMPA-Rs) mediate a majority of excitatory synaptic transmission in the brain. In hippocampus, most AMPA-Rs are hetero-oligomers composed of GluR1/GluR2 or GluR2/GluR3 subunits. Here we show that these AMPA-R forms display different synaptic delivery mechanisms. GluR1/GluR2 receptors are added to synapses during plasticity; this requires interactions between GluR1 and group I PDZ domain proteins. In contrast, GluR2/GluR3 receptors replace existing synaptic receptors continuously; this occurs only at synapses that already have AMPA-Rs and requires interactions by GluR2 with NSF and group II PDZ domain proteins. The combination of regulated addition and continuous replacement of synaptic receptors can stabilize long-term changes in synaptic efficacy and may serve as a general model for how surface receptor number is established and maintained.     

Desensitization by covalent modification of the chemoreceptor of Escherichia coli

Chemoreceptors in Escherichia coli were studied in situ in chemotactic mutants, deficient in the ability to modify the receptors, by using membrane vesicles prepared from the mutants. The affinity of the receptors for the ligands is related to the level of modification of the receptors. Unmodified serine receptor had a dissociation constant of 0.8 microM, while modified receptor had a dissociation constant that was at least 100-times higher. The results are discussed in relation to the two-state model of the chemoreceptor.     

Molecular bases for human complement C7 polymorphisms,C7*3 and C7*4

Complement C7 is one of the components of membrane attack complex (MAC) generated by the terminal complement cascade. C7 protein is polymorphic and most of its polymorphisms have been identified using isoelectric focusing (IEF), which detects protein charge differences. To date, the molecular bases of the polymorphisms detected by IEF have not been determined. In this paper, we describe the structural bases of two C7 IEF-detected polymorphisms, C7*3 and C7*4, both of which are common in Asian populations. C7*3 resulted from substitution of cysteine (Cys) at amino acid residue 106 by charged arginine (Arg; C106R), while charged lysine (Lys) at amino acid residue 398 was replaced by neutral glutamine (Gln; K398Q) in C7*4. As C7*3 is hypomorphic, it is important to study its possible associations with diseases such as immunological disorders and infections. We present genetic bases for this C7 polymorphism, which we determined using polymerase chain reaction (PCR)-based genotyping, a simple and accurate method suitable for large-scale studies.     

Oxygen evolution in dark-developed spruce chloroplasts

Chloroplasts isolated from dark-grown seedlings of Picea abiesshowed activities of DCIP and Fecy photoreductions widiout additionof an electron donor to PS-II. In addition to this, when light-inducedoxygen evolution was measured with an oxygen electrode, a significantamount of oxygen was found. These results indicate that thephotoreductions are coupled to the oxygen-evolving reaction.Furthermore, thylakoid membranes were functional in the protonuptake and the 515-nm absorbance change as parameters of theirphysicochemical functions. Electron microscopy showed that thylakoidswere well-developed with prolamellar bodies and partially stackedto from grana. We conclude that oxygen-evolving ability and the physicochemicalfunction of thylakoid membranes develop in chloroplasts of dark-grownspruce seedlings. (Received September 21, 1974; )