Curcumin and its analogues as potent inhibitors of low density lipoprotein oxidation: H-atom abstraction from the phenolic groups and possible involvement of the 4-hydroxy-3-methoxyphenyl groups

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, 1) is a yellow ingredient isolated from turmeric (Curcumin longa). It has been shown to exhibit a variety of biological activities including antioxidative activity. In order to find more active antioxidants with 1 as the lead compound we synthesized curcumin analogues, i.e., 1,7-bis(3,4-dihydroxyphenyl)-1,6-heptadiene-3,5-dione (2), 1-(3,4-dihydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (3), 1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (4), 1,7-bis (4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (5), 1-(3,4-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (6), 1,7-bis(3,4-dimethoxyphenyl)-1,6- heptadiene-3,5-dione (7), 1,7-bis(4-methoxyphenyl)-1,6-heptadiene-3,5-dione (8), and 1,7-diphenyl-1,6-heptadiene-3,5-dione (9). Antioxidative effects of curcumin and its analogues against free radical initiated peroxidation of human low density lipoprotein (LDL) were studied. The peroxidation was initiated either by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), or by cupric ion (Cu2+). The reaction kinetics were monitored either by the uptake of oxygen and the depletion of alpha-tocopherol present in the native LDL, or by the formation of thiobarbituric acid reactive substances. Kinetic analysis of the antioxidation process demonstrates that these compounds, except 7, 8, and 9, are effective antioxidants against AAPH- and Cu2+ -initiated LDL peroxidation by H-atom abstraction from the phenolic groups. Compounds 2 and 3 which bear ortho-diphenoxyl functionality possess significantly higher antioxidant activity than curcumin and other analogues, and the 4-hydroxy-3-methoxyphenyl group also play an important role in the antioxidative activity.     

Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma

Human intervertebral disc (IVD) degeneration often initiated from the human nucleus pulposus (hNP) with aging leading to IVD destruction and extracellular matrix (ECM) depletion. Previously, we have successfully employed transforming growth factor-beta1 (TGF-beta1) to promote chondrogenesis of mesenchymal progenitor cells (MPCs) and immortalized human mesenchymal stem cells. In this study, we examine the role of TGF-beta1 in platelet-rich plasma (PRP) on disc regeneration, including proliferation, redifferentiation, and the reconstitution of tissue-engineered NP. hNP cells were isolated from volunteers with different ages and cultured in the presence of PRP. We found that the most effective concentration for hNP proliferation was 1 ng/ml TGF-beta1 in PRP, which was further applied in the following experiments. hNP cell proliferation in all age groups were increased time-dependently by PRP and cell morphologies showed aggregation. The mRNA of Sox9, type II collagen, and aggrecan were all significantly upregulated by PRP through RT-PCR. Glycosaminoglycan (GAG) accumulation reached the highest value at day 7 and continued to day 9 culture. PRP promoted NP regeneration via the Smad pathway was also determined and highly activated p-Smad2/3 at 30 min and continuously sustained to 120 min. Immunostaining of type II collagen indicates that PRP participates in chondrogenesis of tissue-engineered NP with collagen scaffolds. We concluded that growth factors in PRP can effectively react as a growth factor cocktail to induce hNP proliferation and differentiation, and also promote tissue-engineered NP formation. These findings are the first to demonstrate that PRP might be a therapeutic candidate for prevention of disc degeneration.     

Overexpression of an Arabidopsis magnesium transport gene, AtMGT1, in Nicotiana benthamiana confers Al tolerance

Aluminium (Al) toxicity is the most important limiting factor for crop production in acid soil environments worldwide. In some plant species, application of magnesium (Mg(2+)) can alleviate Al toxicity. However, it remains unknown whether overexpression of magnesium transport proteins can improve Al tolerance. Here, the role of AtMGT1, a member of the Arabidopsis magnesium transport family involved in Mg(2+) transport, played in Al tolerance in higher plants was investigated. Expression of 35S::AtMGT1 led to various phenotypic alterations in Nicotiana benthamiana plants. Transgenic plants harbouring 35S::AtMGT1 exhibited tolerance to Mg(2+) deficiency. Element assay showed that the contents of Mg, Mn, and Fe in 35S::AtMGT1 plants increased compared with wild-type plants. Root growth experiment revealed that 100 microM AlCl(3) caused a reduction in root elongation by 47% in transgenic lines, whereas root growth in wild-type plants was inhibited completely. Upon Al treatment, representative transgenic lines also showed a much lower callose deposition, an indicator of increased Al tolerance, than wild-type plants. Taken together, the results have demonstrated that overexpression of ATMGT1 encoding a magnesium transport protein can improve tolerance to Al in higher plants.     

RNA-binding proteins SOP-2 and SOR-1 form a novel PcG-like complex in C. elegans

We describe the identification and characterization of a novel PcG gene in C. elegans, sor-1, which is involved in global repression of Hox genes. sor-1 encodes a novel protein with an RNA-binding activity. We provide evidence that SOR-1 and the previously identified RNA-binding protein SOP-2 may constitute an RNA-binding complex in Hox gene repression. SOR-1 and SOP-2 directly interact with each other and are colocalized in nuclear bodies. The localization of SOR-1 depends on SOP-2. Surprisingly, homologs of SOR-1 and SOP-2 are not found in other organisms, including the congeneric species C. briggsae, suggesting an unexpected lack of evolutionary constraint on an essential global gene regulatory system.     

NMPP: a user-customized NimbleGen microarray data processing pipeline

NMPP package is a bundle of user-customized tools based on established algorithms and methods to process self-designed NimbleGen microarray data. It features a command-line-based integrative processing procedure that comprises five major functional components, namely the raw microarray data parsing and integrating module, the array spatial effect smoothing and visualization module, the probe-level multi-array normalization module, the gene expression intensity summarization module and the gene expression status inference module. AVAILABILITY: http://plantgenomics.biology.yale.edu/nmpp     

ATM-Chk2-p53 activation prevents tumorigenesis at an expense of organ homeostasis upon Brca1 deficiency

BRCA1 is a checkpoint and DNA damage repair gene that secures genome integrity. We have previously shown that mice lacking full-length Brca1 (Brca1(delta11/delta11)) die during embryonic development. Haploid loss of p53 completely rescues embryonic lethality, and adult Brca1(delta11/delta11)p53+/- mice display cancer susceptibility and premature aging. Here, we show that reduced expression and/or the absence of Chk2 allow Brca1(delta11/delta11) mice to escape from embryonic lethality. Compared to Brca1(delta11/delta11)p53+/- mice, lifespan of Brca1(delta11/delta11)Chk2-/- mice was remarkably extended. Analysis of Brca1(delta11/delta11)Chk2-/- mice revealed that p53-dependent apoptosis and growth defect caused by Brca1 deficiency are significantly attenuated in rapidly proliferating organs. However, in later life, Brca1(delta11/delta11)Chk2-/- female mice developed multiple tumors. Furthermore, haploid loss of ATM also rescued Brca1 deficiency-associated embryonic lethality and premature aging. Thus, in response to Brca1 deficiency, the activation of the ATM-Chk2-p53 signaling pathway contributes to the suppression of neoplastic transformation, while leading to compromised organismal homeostasis. Our data highlight how accurate maintenance of genomic integrity is critical for the suppression of both aging and malignancy, and provide a further link between aging and cancer.     

Hyperplasia and spontaneous tumor development in the gynecologic system in mice lacking the BRCA1-Delta11 isoform

Alternative splicing in the BRCA1 locus generates multiple protein products including BRCA1-Delta11, which is identical to the BRCA1 full-length isoform (BRCA1-FL) except for the absence of exon 11. Mutation analysis using gene targeting to create null mutations or disrupt BRCA-FL has provided much of our understanding of BRCA1 functions; however, targeted mutation of specific short forms of BRCA1 has not been reported. To understand the physiologic functions of BRCA1-Delta11, we used a knock-in approach that blocks alternative splicing between exons 10 and 12 to prevent the formation of this form of BRCA1. We showed that homozygous mutant mice (Brca1(FL/FL)) were born at a Mendelian ratio without obvious developmental defects. However, the majority of Brca1(FL/FL) female mice showed mammary gland abnormalities and uterine hyperplasia after one year of age with spontaneous tumor formation. Cultured Brca1(FL/FL) cells exhibited abnormal centrosome amplification and reduction of G(1) population that was accompanied by accumulation of cyclin E and cyclin A. Accumulation of cyclin E was also found in epithelial layers of dilated ducts and hyperproliferative lobular regions in the mammary glands of Brca1(FL/FL) mice. These observations provide evidence that BRCA1 splicing variants are involved in BRCA1 functions in modulating G(1)/S transition, centrosome duplication, and repressing tumor formation.     

BRCA1: cell cycle checkpoint, genetic instability, DNA damage response and cancer evolution

Germline mutations of the breast cancer associated gene 1 (BRCA1) predispose women to breast and ovarian cancers. BRCA1 is a large protein with multiple functional domains and interacts with numerous proteins that are involved in many important biological processes/pathways. Mounting evidence indicates that BRCA1 is involved in all phases of the cell cycle and regulates orderly events during cell cycle progression. BRCA1 deficiency, consequently causes abnormalities in the S-phase checkpoint, the G₂/M checkpoint, the spindle checkpoint and centrosome duplication. The genetic instability caused by BRCA1 deficiency, however, also triggers cellular responses to DNA damage that blocks cell proliferation and induces apoptosis. Thus BRCA1 mutant cells cannot develop further into full-grown tumors unless this cellular defense is broken. Functional analysis of BRCA1 in cell cycle checkpoints, genome integrity, DNA damage response (DDR) and tumor evolution should benefit our understanding of the mechanisms underlying BRCA1 associated tumorigenesis, as well as the development of therapeutic approaches for this lethal disease.     

Directed evolution and characterization of Escherichia coli glucosamine synthase

Glucosamine synthase (GlmS) converts fructose-6-phosphate to glucosamine-6-phosphate. Overexpression of GlmS in Escherichia coli increased synthesis of glucosamine-6-P, which was dephosphorylated and secreted as glucosamine into the growth medium. The E. coli glmS gene was improved through error-prone polymerase chain reaction (PCR) in order to develop microbial strains for fermentation production of glucosamine. Mutants producing higher levels of glucosamine were identified by a plate cross-feeding assay and confirmed in shake flask cultures. Over 10 mutants were characterized and all showed significantly reduced sensitivity to inhibition by glucosamine-6-phosphate. Ki of mutants ranged from 1.4 to 4.0 mM as compared to 0.56 mM for the wild type enzyme. Product resistance resulted from single mutations (L468P, G471S) and/or combinations of mutations in the sugar isomerase domain. Most overexpressed GlmS protein was found in the form of inclusion bodies. Cell lysate from mutant 2123-72 contained twice as much soluble GlmS protein and enzyme activity as the strain overexpressing the wild type gene. Using the product-resistant mutant, glucosamine production was increased 60-fold.