9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.     

Ionizing radiation-inducible microRNA miR-193a-3p induces apoptosis by directly targeting Mcl-1

The functions of microRNAs (miRNAs) as either oncogenes or tumor suppressors in regulating cancer-related events have been established. We analyzed the alterations in the miRNA expression profile of the glioma cell line U-251 caused by ionizing radiation (IR) by using an miRNA array and identified several miRNAs whose expression was significantly affected by IR. Among the IR-responsive miRNAs, we further examined the function of miR-193a-3p, which exhibited the most significant growth-inhibiting effect. miR-193a-3p was observed to induce apoptosis in both U-251 and HeLa cells. We also demonstrated that miR-193a-3p induces the accumulation of intracellular reactive oxygen species (ROS) and DNA damage as determined by the level of γH2AX and by performing the comet assay. The induction of both apoptosis and DNA damage by miR-193a-3p was blocked by antioxidant treatment, indicating the crucial role of ROS in the action of miR-193a-3p. Among the putative target proteins, the expression of Mcl-1, an anti-apoptotic Bcl-2 family member, decreased because of miR-193a-3p transfection. A reporter assay using a luciferase construct containing the 3′-untranslated region of Mcl-1 confirmed that Mcl-1 is a direct target of miR-193a-3p. Down-regulation of Mcl-1 by siRNA transfection closely mimicked the outcome of miR-193a-3p transfection showing increased ROS, DNA damage, cytochrome c release, and apoptosis. Ectopic expression of Mcl-1 suppressed the pro-apoptotic action of miR-193a-3p, suggesting that Mcl-1 depletion is critical for miR-193a-3p induced apoptosis. Collectively, our results suggest a novel function for miR-193a-3p and its potential application in cancer therapy.     

Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction.

Protection against ischemic kidney injury is afforded by 24 h of ureteral obstruction (UO) applied 6 or 8 days prior to the ischemia. Uremia or humoral factors are not responsible for the protection, since unilateral UO confers protection on that kidney but not the contralateral kidney. Prior UO results in reduced postischemic outer medullary congestion and leukocyte infiltration. Prior UO results in reduced postischemic phosphorylation of c-Jun N-terminal stress-activated protein kinase 1/2 (JNK1/2), p38, mitogen-activated protein kinase (MAPK) kinase 4 (MKK4), and MKK3/6. Very few cells stain positively for proliferating cell nuclear antigen after obstruction, indicating that subsequent protection against ischemia is not related to proliferation with increased numbers of newly formed daughter cells more resistant to injury. UO increases the expression of heat shock protein (HSP)-25 and HSP-72. The increased HSP-25 expression persists for 6 or 8 days, whereas HSP-72 does not. HSP-25 expression is increased in the proximal tubule cells in the outer stripe of the outer medulla postobstruction, prior to, and 24 h after ischemia. In LLC-PK(1) renal epithelial cells, adenovirus-expressed human HSP-27 confers resistance to chemical anoxia and oxidative stress. Increased HSP-27 expression in LLC-PK(1) cells results in reduced H(2)O(2)-induced phosphorylation of JNK1/2 and p38. In conclusion, prior transient UO renders the kidney resistant to ischemia. This resistance to functional consequences of ischemia is associated with reduced postischemic activation of JNK, p38 MAP kinases, and their upstream MAPK kinases. The persistent increase in HSP-25 that occurs as a result of UO may contribute to the reduction in phosphorylation of MAPKs that have been implicated in adhesion molecule up-regulation and cell death.     

Action of human apurinic endonuclease (Ape1) on C1'-oxidized deoxyribose damage in DNA

Oxidative damage to DNA includes diverse lesions in the sugar-phosphate backbone. The chemical "nuclease" bis(1,10-phenanthroline)copper complex [(OP)(2)Cu] is believed to generate a mixture of direct oxidative strand breaks and C1'-oxidized abasic sites (2-deoxyribonolactone; dL). We found that, under our conditions, the lesions produced by (OP)(2)Cu (50 microM) in synthetic duplex DNA were predominantly dL, accompanied by approximately 30% direct strand breaks with 3'-phosphates. For enzymatic studies, (OP)(2)Cu was used to introduce damage with limited sequence-selectivity, while photolysis of a site-specific 2'-deoxyuridine-1'-t-butyl ketone generated dL at a defined position. The results showed that Ape1, the major human abasic endonuclease, catalyzed 5'-incision of dL sites, but acted at least 10-fold less effectively to remove the 3'-phosphates at direct strand breaks. Kinetic analysis of Ape1 incision using the site-specific dL substrate revealed the same k(cat) for dL and regular (glycosylase-generated) abasic sites, but with K(m) approximately five-fold higher for dL substrate. The efficiency of Ape1 acting on dL, and the abundance of this enzyme in vivo, indicate that dL sites in vivo would be rapidly processed by the endonuclease. The recent observation that Ape1-cleaved dL sites can covalently trap DNA polymerase beta during the abasic excision process suggests that efficient incision of dL by Ape1 may potentiate further problems in DNA repair.     

Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process

A shortcut biological nitrogen removal (SBNR) utilizes the concept of a direct conversion of ammonium to nitrite and then to nitrogen gas. A successful SBNR requires accumulation of nitrite in the system and inhibition of the activity of nitrite oxidizers. A high concentration of free ammonia (FA) inhibits nitrite oxidizers, but unfortunately decreases the ammonium removal rate as well. Therefore, the optimal range of FA concentration is necessary not only to stabilize nitrite accumulation but also to achieve maximum ammonium removal. In order to derive such optimal FA concentrations, the specific substrate utilization rates of ammonium and nitrite oxidizers were measured. The optimal FA concentration range appeared to be 5–10 mg/L for the adapted sludge. The simulated results from the modified inhibition model expressed by FA and ammonium/nitrite concentrations were shown very similar to the experimental results.     

A combinatorial approach to hybrid enzymes independent of DNA homology

We present a methodology, termed incremental truncation for the creation of hybrid enzymes (ITCHY), that creates combinatorial fusion libraries between genes in a manner that is independent of DNA homology. We compared the ability of ITCHY and DNA shuffling to create interspecies fusion libraries between fragments of the Escherichia coli and human glycinamide ribonucleotide transformylase genes, which have only 50% identity on the DNA level. Sequencing of several randomly selected positives from each library illustrated that ITCHY identified a more diverse set of active fusion points including those in regions of nonhomology and those with crossover points that diverged from the sequence alignment. Furthermore, some of the hybrids found by ITCHY that were fused at nonhomologous locations had activities that were greater than or equal to the activity of the hybrids found by DNA shuffling.     

Generation of <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> Ghost by Coexpression of PhiX 174 Lysis <Emphasis Type="Italic">E</Emphasis> gene and Staphylococcal Nuclease <Emphasis Type="Italic">A</Emphasis> Gene

Vibrio anguillarum ghosts (VAG) were generated, for the first time, using a conjugation vector containing a ghost bacteria inducing cassette, pRK-λP_R-cI-Elysis, in which the expression of PhiX174 lysis gene E was controlled by the P _R /cI regulatory system of lambda phage. By scanning electron microscopy, holes ranging 80–200 nm in diameter were observed in the VAG. To avoid the presence of bacterial genomic DNA and an antibiotic resistance gene in the final VAG product, we constructed a new dual vector, pRK-λP_R-cI-E-SNA, containing the E-mediated lysis cassette and the staphylococcal nuclease A (SNA)-mediated DNA degradation cassette, and generated safety-enhanced VAG for use as a fish vaccine.     

Licorice-derived dehydroglyasperin C increases MKP-1 expression and suppresses inflammation-mediated neurodegeneration

Recent studies have demonstrated that microglial hyperactivation-mediated neuroinflammation is involved in the pathogenesis of several neurodegenerative diseases. Thus, inhibiting microglial production of the neurotoxic mediator tumor necrosis factor-α (TNF-α) is considered a promising strategy to protect against neurodegeneration. Here, we investigated the inhibitory effect of licorice-derived dehydroglyasperin C (DGC) on lipopolysaccharide (LPS)-induced TNF-α production and inflammation-mediated neurodegeneration. We found that DGC pre-treatment attenuated TNF-α production in response to LPS stimulation of BV-2 microglia. DGC pre-treatment attenuated LPS-induced inhibitor of κB-α (IκB-α) and p65 phosphorylation and decreased the DNA binding activity of nuclear factor-κB (NF-κB). DGC pre-treatment also inhibited LPS-mediated phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinase (ERK). Interestingly, DGC treatment of BV-2 microglia significantly increased MAPK phosphatase 1 (MKP-1) mRNA and protein expression, which is a phosphatase of p38 MAPK and ERK, suggesting that the DGC-mediated increase in MKP-1 expression might inhibit LPS-induced MAPKs and NF-κB activation and further TNF-α production. We also found that LPS-mediated microglial neurotoxicity can be attenuated by DGC. The addition of conditioned media (CM) from DGC- and LPS-treated microglia to neurons helped maintain healthy cell body and neurite morphology and increased the number of microtubule-associated protein 2-positive cells and the level of synaptophysin compared to treatment with CM from LPS-treated microglia. Taken together, these data suggest that DGC isolated from licorice may inhibit microglia hyperactivation by increasing MKP-1 expression and acting as a potent anti-neurodegenerative agent.