Factors affecting DNA Triplex formation

Triplex-forming oligonucleotides (TFOs) can be used to target DNA in a sequence-specific fashion, and have a number of potential therapeutic and biotechnological applications. TFOs bind within the DNA major groove where they form sequence-specific contacts with exposed groups on the target duplex. Pyrimidine-rich TFOs bind parallel to the target purine strand forming C+.GC and T.AT triplets and usually require conditions of low pH, which are needed for protonation of the third strand cytosines. In contrast, purine-rich TFOs bind antiparallel to the target and form triplexes containing G.GC and A.AT triplets. DNase I footprinting studies with parallel triplexes often reveal enhanced cleavage at the triplex-duplex junction at the 3’-end of the duplex purine strand. This study systematically investigated how this enhanced cleavage is affected by the nature of the base pairs that flank the TFO-binding site. For this we have used the well-characterised TFO-binding site in the tyrT(43-59) fragment and have changed the base at the 3’-end of the homopurine strand from cytosine to each of the other three bases in turn. In each case the footprints were accompanied by enhanced DNase I cleavage at the 3’-triplex-duplex junction on the purine strand, which is thought to be due to local structural changes that render the DNA to be more susceptible to cleavage by the enzyme. The enhancements were generally greater for flanking pyrimidines than purines. Similar experiments investigated the effect of changing the terminal triplet from T.AT to C+.GC, again flanked by each base in turn. Although there were no significant differences in the concentration dependence of the footprints, fluorescence melting experiments showed that triplexes flanked by G and A are more stable than those flanked by C and T. We also used diethylpyrocabonate (DEPC) to probe the reactivity of adenines at the triplex-duplex junction and find that some, but not all, sequence combinations generate enhanced reactivity, suggesting that triplex formation has altered the stacking pattern of adenines on the 3’-side of the TFO binding site. For antiparallel triplex formation, DNase I enhancements were also observed at a number of bands beyond the 5’-end of each TFO’s binding site. This is also attributed to the TFO-induced DNA structural changes that increase the accessibility of the enzyme to the target site. The results of concentration dependence of the footprints are similar to the parallel ones though fragment AC with 17-mer-G TFO had a much lower C50

Young coconut juice accelerates cutaneous wound healing by downregulating macrophage migration inhibitory factor (MIF) in ovariectomized rats: Preliminary novel findings

Estrogens play a crucial role in cutaneous wound healing by down-regulating macrophage migration inhibitory factor (MIF). We had previously reported the effect of young coconut juice (YCJ) known to contain the phytoestrogen, -sitosterol, on cutaneous wound healing in ovariectomized (ovx) rats. This research investigated the possible mechanisms of YCJ on cutaneous wound healing and it was found that it down regulated macrophage migration inhibitory factor (MIF). This resulted in ultrastructural changes that were observed using transmission electron microscopy (TEM). Four groups of female rats (6 in each group) were included in this study: sham-operated, ovariectomized (ovx), ovx that received estradiol benzoate (EB) injections intraperitoneally, and ovx that received YCJ orally. Two weeks after ovariectomy, two equidistant 1-cm full-thickness skin incisional wounds were made. At the end of the third week (7 day treatment) and the fourth week (14 day treatment) of study the rats were sacrificed, and their serum estradiol (E2) levels were measured by a chemiluminescent immunoassay. The skin from the wound was excised and examined by TEM and MIF immunohistochemical staining. The TEM study after 14 days of treatment showed that the size of the keratinocyte cells from the ovx+YCJ group was larger and these cells contained many more cytoplasmic processes than those of the ovx group. The MIF immunoreactivity was also lowest in the ovx+YCJ group. This study showed that there was an increased intercellular exchange via the cytoplasmic processes of the keratinocytes that could account for the promotion of cutaneous wound healing in the ovx rats receiving YCJ, and that the possible mechanism for this was via the down-regulation of MIF

Beneficial effects of young coconut juice feeding on the lipid, renal and liver profiles, in ovariectomized rats: Preliminary novel findings

The purpose of this study was to determine the effects of feeding young coconut juice (YCJ), known to contain - sitosterol, to ovariectomized rats, a model for postmenopausal women, on the lipid, renal and liver metabolism profiles. Four groups of female rats (6 in each group) were included in this study. These included sham-operated, ovariectomized (ovx), ovx receiving estradiol benzoate (EB) injections intraperitoneally, and ovx receiving YCJ orally. At the end of the third and the fourth week of study, the rats were sacrificed and their serum estradiol (E2) was analyzed. The uterus was removed along with the kidney and liver. The latter was paraffin processed for histopathological assessment. In contast with the 7 days treatments, all parameters of the 14 days treatment had improved. After 14 days of treatment, the circulating levels of BUN, creatinine, cholesterol, triglyceride, LDL, AST, ALT, ALP, total protein and albumin of ovx+YCJ group were not significantly different from the sham and ovx groups. Only the serum HDL level of the ovx+YCJ group was significantly higher than that of the sham group. The histopathological assessment of the liver and kidney showed no significant changes when compared with the control groups. Glycogen accumulation appeared in the cytoplasm of the hepatocytes particularly in the ovx+YCJ group but significant changes of the hepatocytes containing glycogen were not detected. No other abnormal features were seen in any of the four groups. The %age uterine/body weights indicated that YCJ feeding in ovx rats at 100 mL/kgBW for up to two weeks did not cause increased uterine weight. In summary, this study confirmed that feeding YCJ had beneficial effects on the serum lipid profile, and maintained liver and renal functions for up to 2 weeks after administration

Ebselen, Iron Uptake Inhibitor, Alleviates Iron Overload-Induced Senescence-Like Neuronal Cells SH-SY5Y via Suppressing the mTORC1 Signaling Pathway

Increasing evidence highlights that excessive iron accumulation in the brain plays a vital role in neuronal senescence and is implicated in the pathogenesis of age-related neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Therefore, the chemical compounds that eliminate an iron overload may provide better protection against oxidative stress conditions that cause the accumulation of senescent cells during brain aging. Ebselen has been identified as a strongly useful compound in the research on redox biology mechanisms. We hypothesized that ebselen could alleviate an iron overload-induced oxidative stress and consequently reverses the senescence-like phenotypes in the neuronal cells. In the present study, SH-SY5Y cells were treated with ferric ammonium citrate (FAC) before ebselen, and the evaluation of the cellular iron homeostasis, the indicators of oxidative stress, and the onset of senescence phenotypes and mechanisms were carried out accordingly. Our findings showed that ebselen ameliorated the FAC-mediated iron overload by decreasing the expression of divalent metal transporter 1 (DMT1) and ferritin light chain (FT-L) proteins. In contrast, it increased the expression of ferroportin 1 (FPN1) protein and its correlation led to a decrease in the expression of the cytosolic labile iron pool (LIP). Furthermore, ebselen significantly reduced reactive oxygen species (ROS) and rescued the mitochondrial membrane potential (ΔΨm). Notably, ebselen restored the biomarkers of cellular senescence by reducing the number of senescence-associated β-galactosidase (SA-β-gal) positive cells and senescence-associated secretory phenotypes (SASP). This also suppressed the expression of p53 protein targeting DNA damage response (DDR)/p21 cyclin-dependent kinase (CDK) inhibitor through a mTORC1 signaling pathway. Potentially, ebselen could be a therapeutic agent for treating brain aging and AD by mitigating iron accumulation and restoring senescence in SH-SY5Y cells

DNA structural changes induced by intermolecular triple helix formation

DNase I footprints of intermolecular DNA triplexes are often accompanied by enhanced cleavage at the 3′-end of the target site at the triplex–duplex junction. We have systematically studied the sequence dependence of this effect by examining oligonucleotide binding to sites flanked by each base in turn. For complexes with a terminal T.AT triplet, the greatest enhancement is seen with ApC, followed by ApG and ApT, with the weakest enhancement at ApA. Similar DNase I enhancements were observed for a triplex with a terminal C+.GC triplet, though with little difference between the different GpN sites. Enhanced reactivity to diethylpyrocarbonate was observed at As that flank the triplex–duplex junction at AAA or AAC but not AAG or AAT. Fluorescence melting experiments demonstrated that the flanking base affected the stability with a 4 °C difference in Tm between a flanking C and G. Sequences that produced the strongest enhancement correlated with those having the lower thermal stability. These results are interpreted in terms of oligonucleotide-induced changes in DNA structure and/or flexibility

Young coconut juice can accelerate the healing process of cutaneous wounds

<p>Abstract</p> <p>Background</p> <p>Estrogen has been reported to accelerate cutaneous wound healing. This research studies the effect of young coconut juice (YCJ), presumably containing estrogen-like substances, on cutaneous wound healing in ovairectomized rats.</p> <p>Methods</p> <p>Four groups of female rats (6 in each group) were included in this study. These included sham-operated, ovariectomized (ovx), ovx receiving estradiol benzoate (EB) injections intraperitoneally, and ovx receiving YCJ orally. Two equidistant 1-cm full-thickness skin incisional wounds were made two weeks after ovariectomy. The rats were sacrificed at the end of the third and the fourth week of the study, and their serum estradiol (E2) level was measured by chemiluminescent immunoassay. The skin was excised and examined in histological sections stained with H&E, and immunostained using anti-estrogen receptor (ER-α an ER-β) antibodies.</p> <p>Results</p> <p>Wound healing was accelerated in ovx rats receiving YCJ, as compared to controls. This was associated with significantly higher density of immunostaining for ER-α an ER-β in keratinocytes, fibroblasts, white blood cells, fat cells, sebaceous gland, skeletal muscles, and hair shafts and follicles. This was also associated with thicker epidermis and dermis, but with thinner hypodermis. In addition, the number and size of immunoreactive hair follicles for both ER-α and ER-β were the highest in the ovx+YCJ group, as compared to the ovx+EB group.</p> <p>Conclusions</p> <p>This study demonstrates that YCJ has estrogen-like characteristics, which in turn seem to have beneficial effects on cutaneous wound healing.</p