The acetyl-TESHHK-amide peptide, modeling a part of the C-terminal "tail" of histone H2A, was found previously by us to undergo at pH 7. 4 a Ni(II)-assisted hydrolysis of the E-S peptide bond with formation of a stronger Ni(II) complex with the SHHK-amide product [Bal, W., et al. (1998) Chem. Res. Toxicol. 11, 1014-1023]. To further characterize the hydrolysis and test the resulting Ni(II) complex for redox activity, bovine histone H2A and three peptides were investigated: acetyl-LLGKVTIAQGGVLPNIQAVLLPKKTESHHKAKGK (H2A(34)), modeling the entire "C-tail" of H2A; SHHKAKGK (H2A(8)), modeling the cutoff product of hydrolysis; and acetyl-KTESHKAKGK (H2A(10)), modeling a putative Ni(II) binding site in a minor variant H2A.4 of human histone H2A. The Ni(II)-assisted hydrolysis of H2A and H2A(34) was found to proceed approximately 7-fold faster than that of the Ni(II)-acetyl-TESHHK-amide complex under comparable conditions. In both cases, the Ni(II) complex with H2A(8) was the smaller product of the hydrolysis, indicating a high site specificity of the reaction. Of three other metals tested with H2A(34), only Cu(II) cleaved the E-S bond, although much less efficiently than Ni(II); Co(II) and Zn(II) had no effect whatsoever. The H2A(10) peptide appeared to be fully resistant to hydrolytic cleavage and did not exhibit any redox activity versus H(2)O(2) in the presence of Ni(II) at pH 7.4. Likewise, redox-inactive was the Ni(II)-H2A(34) complex. In contrast, the Ni(II)-H2A(8) complex promoted oxidative damage of pUC19 DNA by H(2)O(2), evidenced by a significant increase in the number of single strand breaks and nucleobase modifications typical for a hydroxyl radical-like species attack on DNA. Interestingly, instead of 8-oxopurines, the corresponding formamidopyrimidines were the major products of the damage. The difference in redox activity between the Ni(II)-H2A(34) and Ni(II)-H2A(8) complexes is most likely associated with their different geometries: octahedral and square planar, respectively. Incubation of the Ni(II)-H2A(8) complex with H(2)O(2) also resulted in degradation of the peptide ligand, especially at its Ser and His residues. Thus, binding of Ni(II) to the ESHHK motif of the histone H2A C-tail is damaging to the histone C-terminal tail and to histone-associated DNA. The results support a dual mechanism of Ni(II)-induced carcinogenesis, including both genotoxic and epigenetic effects.
Withaferin A, a major chemical constituent of Withania somnifera, has been reported for its tumor cell growth inhibitory activity, antitumor effects, and impairing metastasis and angiogenesis. The mechanism by which withaferin A initiates apoptosis remains poorly understood. In the present report, we investigated the effect of withaferin A on the apoptotic pathway in U937 human promonocytic cells. We show that withaferin A induces apoptosis in association with the activation of caspase-3. JNK and Akt signal pathways play crucial roles in withaferin A-induced apoptosis in U937 cells. Furthermore, we have shown that overexpression of Bcl-2 and active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase-3, and PLC-gamma1 cleavage by withaferin A. Taken together, our results indicated that the JNK and Akt pathways and inhibition of NF-kappaB activity were key regulators of apoptosis in response to withaferin A in human leukemia U937 cells.
The human tooth is the hardest organ of the body, and is composed of enamel, dentin, and dental pulp. Dentin provides the basis of the tooth shape by lining the inner parts of the root and crown. Odontoblasts deposit dentin, an organic matrix that contains collagen, noncollagenous proteins, phospholipids, and growth factors. In this study, we sought to reveal the proteins in human dentin by using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic approaches. Human third molar dentins were cut, isolated, and demineralized, and the extracted proteins were separated on SDS-PAGE. In-gel digested peptides were analyzed using reverse-phase LC-MS/MS. We identified 233 total and 68 common proteins from 3 individuals with high confidence, including a variety of collagenous and noncollagenous proteins such as DSPP, biglycan, osteoglycin, osteopontin, and osteocalcin. In addition to known proteins, we also identified various matrix and serum proteins deposited in the dentin, including asporin, lumican, mimecan, and SOD3. This study provides the first list of proteomes that are detected in human dentin. This proteome list is useful in that it defines the organic matrix of dentin and helps to characterize odontoblasts.
Genistein is an isoflavone that is known to be contained in soybean. It was proved that genistein plays a pivotal role in homeostasis in the human body. In the course of screening for useful K K-glucosidase inhibitors, we isolated and identified genistein as a candidate for K K-glucosidase inhibitor from fermentation broths of a Streptomyces sp. Genistein was shown to be a reversible, slow-binding, non-competitive inhibitor of yeast K K-glucosidase with a K i value of 5.7U U10 38 M when the enzyme mixture was pretreated with genistein. These results show a possibility that genistein could be a useful tool for metabolic disorders. ß
The development of antimelanogenic agents is important for the prevention of serious aesthetic problems such as melasma, freckles, age spots and chloasma. The aim of this study was to investigate the antimelanogenic effect of sesamol, an active lignan isolated from Sesamum indicum, in melan‐a cells. Sesamol strongly inhibited melanin biosynthesis and the activity of intracellular tyrosinase by decreasing cyclic adenosine monophosphate (cAMP) accumulation. Sesamol significantly decreased the expression of melanogenesis‐related genes, such as tyrosinase, tyrosinase‐related protein‐1,2 (TRP‐1,2), microphthalmia‐associated transcription factor (MITF) and melanocortin 1 receptor (MC1R). In addition, sesamol also induces phosphorylation of p38 mitogen‐activated protein kinase (p38 MAPK) and c‐Jun N‐terminal kinase (JNK). Moreover, sesamol dose‐dependently decreased zebrafish pigment formation, tyrosinase activity and expression of melanogenesis‐related genes. These findings indicate that sesamol inhibited melanin biosynthesis by down‐regulating tyrosinase activity and melanin production via regulation of gene expression of melanogenesis‐related proteins through modulation of MITF activity, which promoted phosphorylation of p38 and JNK in melan‐a cells. Together, these results suggest that sesamol strongly inhibits melanin biosynthesis, and therefore, sesamol represents a new skin‐whitening agent for use in cosmetics.
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