Structure-activity relationship of N-methyl-bisindolylmaleimide derivatives as cell death inhibitors

A series of N-methyl-bisindolylmaleimide derivatives was synthesized and evaluated as cell death inhibitors. N-Methyl-2-[1-(3-aminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide (21) was the most potent inhibitor of H2O2-induced necrotic death of human leukemia HL60 cells among them.     

Efficient cleavage of RNA, enhanced cellular uptake, and controlled intracellular localization of conjugate DNAzymes

Conjugate DNAzymes with polyamines and peptides were successfully prepared by solid phase fragment condensation (SPFC) and showed up to 4.2 times higher catalytic efficiency (k(cat)/K(m)) and enhanced tolerance against DNase 1digestion. To be pointed out, intracellular localization of DNAzymes could be controlled by conjugated with naturally occurring signal peptides responsible for nuclear cytoplasmic transport of proteins.     

Corynebacterium sp. U-96 contains a cluster of genes of enzymes for the catabolism of sarcosine to pyruvate

The sarcosine oxidase gene and nearby genes from Corynebacterium sp. U-96 were determined. The genes for serine hydroxymethyltransferase, the beta, delta, alpha, and gamma subunits of sarcosine oxidase, serine dehydratase, and 10-formyltetrahydrofolate hydrolase are arranged in this order. This suggests that the bacteria contain a cluster of genes for the catabolism of sarcosine to pyruvate. The possibility that the gene cluster is a merit for the cellular energy demands of the bacteria is discussed. Functional expression of sarcosine oxidase in Escherichia coli was accomplished, but the beta subunit and the betadelta complex were expressed at a low level as a soluble protein.     

Involvement of MEK-ERK signaling pathway in the inhibition of cell growth by troglitazone in human pancreatic cancer cells

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.     

Multiple N-cadherin enhancers identified by systematic functional screening indicate its Group B1 SOX-dependent regulation in neural and placodal development

Neural plate and sensory placodes share the expression of N-cadherin and Group B1 Sox genes, represented by Sox2. A 219-kb region of the chicken genome centered by the N-cadherin gene was scanned for neural and placodal enhancers. Random subfragments of 4.5 kb average length were prepared and inserted into tkEGFP reporter vector to construct a library with threefold coverage of the region. Each clone was then transfected into N-cadherin-positive (lens, retina and forebrain) or -negative embryonic cells, or electroporated into early chicken embryos to examine enhancer activity. Enhancers 1-4 active in the CNS/placode derivatives and non-specific Enhancer 5 were identified by transfection, while electroporation of early embryos confirmed enhancers 2-4 as having activity in the early CNS and/or sensory placodes but with unique spatiotemporal specificities. Enhancers 2-4 are dependent on SOX-binding sites, and misexpression of Group B1 Sox genes in the head ectoderm caused ectopic development of placodes expressing N-cadherin, indicating the involvement of Group B1 Sox functions in N-cadherin regulation. Enhancers 1, 2 and 4 correspond to sequence blocks conserved between the chicken and mammalian genomes, but enhancers 3 and 5 are unique to the chicken.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.     

Ultrasonication-induced amyloid fibril formation of beta2-microglobulin

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.     

Critical balance of electrostatic and hydrophobic interactions is required for beta 2-microglobulin amyloid fibril growth and stability

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.     

Synthesis and enzyme-catalyzed hydrolysis of a radical-masked glycosylated spin-label reagent

N1-Acetoxy-2,2,6,6-tetramethylpiperidin-4-yl 2,3,4,6-tetra-O-benzyl-alpha- and -beta-D-glucopyranosides (3-alpha, beta) and N1-acetoxy-2,2,5,5-tetramethylpyrrolin-3-oyl 2,3,4,6-tetra-O-benzyl-alpha- and -beta-D-glucopyranosylamines (9-alpha, beta) were synthesized in good yield by Schmidt's glycosylation method. Their subsequent O-debenzylation was proceeded successfully to give the desired products 1-alpha, and 1-beta in good yield, and 2-alpha in a low yield, without 2-beta by only short-timed hydrogenolysis in the presence of palladium-on-carbon (Pd-C) in a CHCl3-MeOH solvent system that included concentrated HCl. Upon enzyme-catalyzed hydrolysis, only 2-alpha was hydrolyzed by the esterase, while both of 1-alpha and 1-beta were not hydrolyzed by any other enzyme such as lipase. These 2-alpha can likely be used as a new water-soluble radical-masked glycosylated spin-label reagent.