Accelerated chronological aging of a mutant fission yeast deficient in both glutathione and superoxide dismutase having cu and zn as cofactors and its enhancement by sir2 deficiency

A mutant of Schizosaccharomyces pombe deficient in both superoxide dismutase with copper and zinc as cofactors and glutathione was hypersensitive to menadione, which intracellularly generates superoxide radicals, and showed short chronological lifespan with more oxidation of proteins. Disruption of the sir2 gene in the double mutant enhanced the short chronological lifespan without more enhanced protein oxidation.     

Unexpected Mechanism of Symbiont-Induced Reversal of Insect Sex: Feminizing Wolbachia Continuously Acts on the Butterfly Eurema hecabe during Larval Development

When the butterfly Eurema hecabe is infected with two different strains (wHecCI2 and wHecFem2) of the bacterial endosymbiont Wolbachia, genetic males are transformed into functional females, resulting in production of all-female broods. In an attempt to understand how and when the Wolbachia endosymbiont feminizes genetically male insects, larval insects were fed an antibiotic-containing diet beginning at different developmental stages until pupation. When the adult insects emerged, strikingly, many of them exhibited sexually intermediate traits in their wings, reproductive organs, and genitalia. The expression of intersexual phenotypes was strong in the insects treated from first instar, moderate in the insects treated from third instar, and weak in the insects treated from fourth instar. The insects treated from early larval instar grew and pupated normally but frequently failed to emerge and died in the pupal case. The dead insects in the pupal case contained lower densities of the feminizing Wolbachia endosymbiont than the successfully emerged insects, although none of them were completely cured of the symbiont infection. These results suggest the following: (i) the antibiotic treatment suppressed the population of feminizing Wolbachia endosymbionts; (ii) the suppression probably resulted in attenuated feminizing activity of the symbiont, leading to expression of intersexual host traits; (iii) many of the insects suffered pupal mortality, possibly due to either intersexual defects or Wolbachia-mediated addiction; and hence (iv) the feminizing Wolbachia endosymbiont continuously acts on the host insects during larval development for expression of female phenotypes under a male genotype. Our finding may prompt reconsideration of the notion that Wolbachia-induced reproductive manipulations are already complete before the early embryonic stage and provide insights into the mechanism underlying the symbiont-induced reversal of insect sex.     

Secretion of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from cultured chick embryo chondrocytes.

We found that chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase were released into the culture medium from the cultured chick embryo chondrocytes. Since the release of the sulfotransferases was observed not only in serum-supplemented medium but also in serum-free medium, the released sulfotransferases were unlikely to be derived from serum. Addition of ascorbate to the serum-free medium supported the continuous release of the sulfotransferases. Monensin, which is known to cause dilatation of the Golgi apparatus and to inhibit sulfation of proteoglycan, was found to affect the release of the sulfotransferases. In the presence of 10(-6) M monensin, chondroitin 6-sulfotransferase activity in the cell layer was decreased to less than one tenth of the control, and the rate of the release of the activity became much smaller than the control after the initial rapid release. The activity of chondroitin 4-sulfotransferase was also affected by monensin, but the reduction of the chondroitin 4-sulfotransferase activity in the cell layer was not so great as the reduction of chondroitin 6-sulfotransferase activity. Unlike to the microsomal sulfotransferases, both chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase released into the culture medium were retained in the soluble fraction after centrifugation at 100,000 x g for 60 min, and were not activated by detergent. pH optimum and requirements for sulfhydryl compounds of the released sulfotransferases were similar to those observed previously in the chondroitin sulfotransferases from chick embryo cartilage and from cultured chick embryo chondrocytes. These results suggest that chondroitin sulfotransferases, which are localized in the Golgi apparatus, may be secreted to the extracellular space in a soluble form under the culture conditions.     

Purification and characterization of a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051; application to the recovery of bioactive peptides from fusion proteins by sequence-specific digestion

Screening cultures of nonpathogenic microorganisms led us to a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051, which we purified and named BSase. The nucleotide sequence encoding BSase, with a molecular mass of 23 894 Da, completely agreed with that of the mpr gene, which had been reported by Rufo Jr. and Sloma et al. to encode a metalloprotease [J Bacteriol (1990) 172:1019–1023 and 1024–1029 respectively]. However, enzymatic characterization revealed it to have the catalytic triad of a serine protease and not the consensus sequence of a metalloprotease, and it was inhibited by diisopropylfluorophosphate. We therefore consider BSase (mpr) to be a serine protease. In the alignment of the acidic-amino-acid-specific proteases, the proteases from bacilli have a highly conserved histidine residue, which is most important in the histidine triad in the proteases from streptomycetes. Furthermore, Ca²⁺ was necessary for its activity and stability. BSase cleaved the C-terminal glutamic acid with high specificity and was very stable over a wide pH range. On the basis of these properties, we tried to retrieve a bioactive peptide from a fusion protein by sequence-specific digestion, and succeeded in obtaining the bioactive peptide. BSase was found to be very useful as a tool for selective cleavage. Received: 24 December 1996 / Received revision: 3 February 1997 / Accepted: 22 February 1997     

Cloning of the structural gene for Clostridium botulinum type C1 toxin and whole nucleotide sequence of its light chain component

The toxigenicity of Clostridium botulinum type C1 is mediated by specific bacteriophages. DNA was extracted from one of these phages. Two DNA fragments, 3 and 7.8 kb, which produced the protein reacting with antitoxin serum were cloned by using bacteriophage lambda gt11 and Escherichia coli. Both DNA fragments were then subcloned into pUC118 plasmids and transferred into E. coli cells. The nucleotide sequences of the cloned DNA fragments were analyzed by the dideoxy chain termination method, and their gene products were analyzed by Western immunoblot. The 7.8-kb fragment coded for the entire light chain component and the N terminus of the heavy chain component of the toxin, whereas the 3-kb fragment coded for the remaining heavy chain component. The entire nucleotide sequence for the light chain component was determined, and the derived amino acid sequence was compared with that of tetanus toxin. It was found that the light chain component of C1 toxin possessed several amino acid regions, in addition to the N terminus, that were homologous to tetanus toxin.     

Discovery and optimization of potent and selective triazolopyridazine series of c-Met inhibitors

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. We previously showed that O-linked triazolopyridazines can be potent inhibitors of c-Met. Herein, we report the discovery of a related series of N-linked triazolopyridazines which demonstrate nanomolar inhibition of c-Met kinase activity and display improved pharmacodynamic profiles. Specifically, the potent time-dependent inhibition of cytochrome P450 associated with the O-linked triazolopyridazines has been eliminated within this novel series of inhibitors. N-linked triazolopyridazine 24 exhibited favorable pharmacokinetics and displayed potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver PD model. Once-daily oral administration of 24 for 22 days showed significant tumor growth inhibition in an NIH-3T3/TPR-Met xenograft mouse efficacy model.     

A conceptual model of the polyamine binding site of N1-acetylpolyamine oxidase developed from a study of polyamine derivatives

We used various polyamine derivatives to study the substrate binding site of N ¹-acetylpolyamine oxidase (PAO) that was partially purified from rat liver. The substrate activities of acetylpolyamines indicated the presence of two anionic centers corresponding to the 1,3-diaminopropane (1,3-DAP) structure and a hydrophobic region in addition to the cleavage site of the acetamidopropyl group. Based on the results of the inhibitory activities of 1,3-DAP derivatives, we developed a conceptual model of the polyamine binding site of PAO. We used this model to identify a potent competitive inhibitor, N ¹,N ⁷-dihexyl-1,7-diamino-4-azaheptane, and to develop an affinity column, 1,16-diamino4,13-diazahexadecane–linked Sepharose, which was useful for the purification of PAO.     

The Death Effector Domain-containing DEDD Supports S6K1 Activity via Preventing Cdk1-dependent Inhibitory Phosphorylation

Cell cycle regulation and biochemical responses upon nutrients and growth factors are the major regulatory mechanisms for cell sizing in mammals. Recently, we identified that the death effector domain-containing DEDD impedes mitotic progression by inhibiting Cdk1 (cyclin-dependent kinase 1) and thus maintains an increase of cell size during the mitotic phase. Here we found that DEDD also associates with S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase, and supports its activity by preventing inhibitory phosphorylation of S6K1 brought about by Cdk1 during the mitotic phase. DEDD^-/- cells showed reduced S6K1 activity, consistently demonstrating decreased levels in activating phosphorylation at the Thr-389 site. In addition, levels of Cdk1-dependent inhibitory phosphorylation at the C terminus of S6K1 were enhanced in DEDD^-/- cells and tissues. Consequently, as in S6K1^-/- mice, the insulin mass within pancreatic islets was reduced in DEDD^-/- mice, resulting in glucose intolerance. These findings suggest a novel cell sizing mechanism achieved by DEDD through the maintenance of S6K1 activity prior to cell division. Our results also suggest that DEDD may harbor important roles in glucose homeostasis and that its deficiency might be involved in the pathogenesis of type 2 diabetes mellitus.Cell size is closely related to specialized cell function and to the specific patterning of tissues in the body. Cell sizing is regulated mainly by two mechanisms: cell cycle control and the biochemical response to nutrients and/or growth factors (1–5). During cell cycle progression, both the G₁ (which is believed to be dominant) and the G₂ periods are important for cells to increase their volume (6–9). In addition, we recently provided evidence that the mitotic period (M phase) also influences cell size, through analysis of DEDD-deficient mice (10, 11). The DEDD molecule was initially described as a member of the death effector domain (DED)²-containing protein family (12). Although the absence of DEDD did not apparently influence progression of apoptosis (10), we found that during mitosis, DEDD is associated with Cdk1-cyclin B1 and that it decreases the kinase activity of Cdk1. This response impedes the Cdk1-dependent mitotic program to shut off synthesis of ribosomal RNA (rRNA) and protein and is consequently useful in gaining sufficient cell growth prior to cell division. Depletion of DEDD consistently results in a shortened mitotic duration and an overall reduction in the amount of cellular rRNA and protein and, furthermore, in cell and body size (10, 11).Of the biochemical responses responsible for cell sizing, the signaling cascade involving phosphatydilinositol 3-kinase (PI3K) and its downstream target of rapamycin (TOR) is most crucial (13–15). In mammals, upon stimulation by growth factors, including insulin, the mammalian TOR (mTOR) cooperates with PI3K-dependent effectors to activate S6K1, thereby phosphorylating the 40 S ribosomal protein S6, and subsequently enhances translation of the 5′-terminal oligopyrimidine sequences that encode components of the translational machinery. This reaction increases the number of ribosomes and the efficacy of protein synthesis, thus critically promoting cell growth (16–18). Therefore, mice deficient for S6K1 (S6K1^-/-) had reduced cell and body size (19–23). This effect also involves S6K1 in maintenance of glucose tolerance. S6K1 significantly supports the size of insulin-producing β cells within pancreatic Langerhans islets (24, 25). Thus, in S6K1^-/- mice, the insulin mass was diminished, which resulted in ineffective secretion of insulin upon glucose administration (21, 23).The activation of S6K1 proceeds through chronological phosphorylation at various residues, toward the crucial phosphorylation of Thr-389, present within the linker domain between the catalytic domain and the carboxyl tail, to obtain maximal enzymatic activity (26). Interestingly, phosphorylation at several Ser/Thr residues within the C-terminal autoinhibitory tail appears to either activate or inhibit S6K1, depending on the cell cycle phase. Shah et al. (27) demonstrated that phosphorylation of those residues (featured by the Thr-421/Ser-424 site) during mitosis pursued by Cdk1 inactivates S6K1 to terminate protein synthesis prior to cell division (28). A recent report by Schmidt et al. (29) demonstrating that phosphorylation of Thr-421/Ser-424 is specifically increased during the G₂/M phase may also support the finding, whereas during the G₁ phase, there is consensus that the phosphorylation at the autoinhibitory domain is requisite for S6K1 activation (26), as also recently demonstrated by Hou et al. (30), where the Cdk5 phosphorylates the Ser-411 site, leading to activation of S6K1. In contrast to such inhibitory regulation of S6K1 during mitosis, however, a recent report by Boyer et al. (31) sharply demonstrated that the activity of S6K1 peaks at mitosis, suggesting that S6K1 may also have some roles during the mitotic phase. If so, how its activity is supported against the inhibitory regulation caused by Cdk1 remains an open question.Hence, the two observations above that both DEDD^-/- and S6K1^-/- situations decrease the efficacy of ribosome and protein synthesis, resulting in smaller cell and body size, and that mitotic Cdk1 has a functional interaction with both S6K1 and DEDD led us here to assess a possible role of DEDD in the context of the functional regulation of S6K1.