Relation between D-glucose and L- and D-alanine in the initiation of germination of Bacillus subtilis spore

The rate of L-alanine-initiated germination of Bacillus subtilis spore was measured by both loss of heat resistance and loss of turbidity, and the effect of glucose on the germination response to a wide range of concentrations of the germinant was analyzed in the presence and absence of D-alanine, an inhibitor. Glucose stimulated L-alanine germination by means of a cooperative effect: glucose increased the affinity of L-alanine by about 3-fold and the rate of germination by about 1.3-fold. However, glucose had little effect on the binding affinity of D-alanine. The apparent binding constant of L-alanine to the spore, which was determined by the next measurable event in the trigger reaction, was 1.2 X 10(-5), that of D-alanine was 6 X 10(-6), and that of glucose was 5 X 10(-5). The relation between the binding site for glucose and those for L- and D-alanine on the spore is discussed. Effect of glucose analogs was also examined.     

Analysis of the mode of antigen presentation required for triggering of T cell proliferation by using various azobenzenearsonate-tyrosine derivatives

Various azobenzenearsonate-tyrosine (ABA-Tyr) derivatives were synthesized by modifying amino and carboxyl groups at the alpha-carbon of tyrosine, with preservation of most of the ABA-Tyr moiety (ABA plus hydroxyphenyl portion of tyrosine). These derivatives were tested for the ability to stimulate ABA-L-Tyr specific T cell lines derived from B10.BR and B10.S mice. ABA-acetyltyramine, ABA-hydroxyphenylpropionic acid (ABA-PPr), and ABA-propylphenol, which lack either the carboxyl or amino group or both, could not induce T cell proliferation. The lack of stimulation by these derivatives was not due to their cytotoxic effects. A similar pattern of proliferation was obtained on stimulating lymph node T cells from B10.BR and B10.S mice primed with ABA-L-Tyr. Some differences were observed, however, between B10.BR and B10.S mice. ABA-L-Tyr-specific T cells from B10.BR mice could not respond well to ABA-D-Tyr in contrast to B10.S T cells. Furthermore, B10.BR mice primed with ABA-acetyltyramine or ABA-PPr in complete Freund's adjuvant could not induce ABA-L-Tyr-reactive T cells, whereas T cells from B10.S mice primed with these derivatives could proliferate in the presence of ABA-L-Tyr. The differences between B10.BR and B10.S mice were further investigated by using (B10.S X B10.BR)F1 mice. T cells from ABA-L-Tyr-immunized F1 mice responded poorly to ABA-D-Tyr when presented with B10.BR antigen-presenting cells (APC), but responded well when presented with B10.S APC. Similarly, T cells from ABA-PPr-primed F1 mice did not proliferate to ABA-L-Tyr in the presence of B10.BR APC, but could proliferate in the presence of B10.S APC. Our results clearly indicate that the presence of charged groups at the alpha-carbon of tyrosine plays a critical role in the triggering of ABA-L-Tyr-specific T cell proliferation. The significance of these results is discussed.     

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCα^S657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCα^S657 and FoxO1.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Use of an activated carbon column for the synthesis of disaccharides by use of a reversed hydrolysis activity of β-galactosidase

Summary An activated carbon column was utilized for the synthesis of disaccharides by use of a reversed hydrolysis activity of an immobilized -galactosidase column in order to shift the equilibrium to the direction of condensation. The yields of lactulose and allo-lactulose from galactose and fructose, and N-acetyl lactosamine and N-acetyl allolactosamine from galactose and N-acetyl glucosamine, were 11.3% and 10.0%, respectively.     

Separation of Cl-Containing Chlorophylls by Column Chromatography

Using Toyopearl and cyclohexane: cyclohexanol solvent, fourCl-containing Chls were separated from ³⁶Cl-labeled cells ofthe blue-green, Plectonema boryanum. In normally grown cells,all four Cl-containing chlorophylls amounted to less than 1/2,000of the total Chi and about 1/50 of P700, values much lower thanpreviously reportedcontents of Chi RC I, and varied from algato alga. The level of Cl-containing Chi was markedly enhancedwhen the cells were poisoned with methyl viologen. These resultssuggests that these Cl-containing Chls are not related to thereaction center of PS I. (Received June 23, 1987; Accepted September 17, 1987)     

In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract

An in vitro splicing system was constructed using portions of chicken delta-crystallin pre-mRNA synthesized in vitro and a HeLa nuclear extract. Analysis of the reaction products revealed that about 25% of the pre-mRNA was precisely spliced at 30 degrees C in 2 h under the standard conditions. The other major products of the reaction detected were a 5'-exon fragment and three RNA species showing unusual electrophoretic mobilities on polyacrylamide gels. Structural analyses showed that these three RNAs contain a branch (lariat) structure as seen in the in vitro splicing reactions of human beta-globin, adenovirus, and yeast pre-mRNAs. In addition, methylation at the N-7 position of the blocking guanosine of the 5'-terminal cap structure of pre-mRNA has been suggested to play an important role in the splicing reaction.