Effects of pergolide mesylate on transduction efficiency of PEP-1-catalase protein

The low transduction efficiency of various proteins is an obstacle to their therapeutic application. However, protein transduction domains (PTDs) are well-known for a highly effective tool for exogenous protein delivery to cells. We examined the effects of pergolide mesylate (PM) on the transduction of PEP-1-catalase into HaCaT human keratinocytes and mice skin and on the anti-inflammatory activity of PEP-1-catatase against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation using Western blot and histological analysis. PM enhanced the time- and dose-dependent transduction of PEP-1-catalase into HaCaT cells without affecting the cellular toxicity. In a mouse edema model, PEP-1-catalase inhibited the increased expressions of inflammatory mediators and cytokines such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1β, and tumor necrosis factor-α induced by TPA. On the other hand, PM alone failed to exert any significant anti-inflammatory effects. However, the anti-inflammatory effect of co-treatment with PEP-1-catalase and PM was more potent than that of PEP-1-catalase alone. Our results indicate that PM may enhance the delivery of PTDs fusion therapeutic proteins to target cells and tissues and has potential to increase their therapeutic effects of such drugs against various diseases.     

Epigenetic mechanism of tumor promotion. Interrelationship between inflammation and ornithine decarboxylase activity induced in vitro by the carcinogenic 12-O-tetradecanoyl-phorbol-13-acetate

Topical application of 2 micrograms 12-O-tetradecanoyl-phorbol-13-acetate (TPA) regularly induced two early events in mouse skin: inflammatory reaction localized in dermal compartment and stimulation of ornithine decarboxylase activity in epidermal cells, in relation to polyamine synthesis and cell division. These reactions were followed, after 48 hrs. by an epidermal hyperplasia. At this stage, another TPA treatment induced a strong ODC activity concurrent with severe inflammation of the dermis. Inhibition of the synthesis of inflammatory factors may antagonize TPA-induced ODC, but the protective potencies differs according to the evolutive stages of the cell. After the first TPA treatment the anti-inflammatory compounds dexamethasone and indomethacin effectively inhibited ODC activity. In contrast during the proliferative stage epidermal cell function may be less dependent on inflammatory factors; thus only partial protection was observed with the inflammatory inhibitors.     

Enhancement of anti-inflammatory activity of PEP-1-FK506 binding protein by silk fibroin peptide

Silk fibroin (SF) peptide has been traditionally used as a treatment for flatulence, spasms, and phlegm. In this study, we examined whether SF peptide enhanced the antiinflammatory effect of PEP-1-FK506 binding protein (PEP-1-FK506BP) through comparing the anti-inflammatory activities of SF peptide and/or PEP-1-FK506BP. In the presence or absence of SF peptide, transduction levels of PEP-1-FK506BP into HaCaT cells and mice skin and anti-inflammatory activities of PEP-1-FK506BP were identified by Western blot and histological analyses. SF peptide alone effectively reduced both mice ear edema and the elevated levels of cyclooxygenase-2, interleukin-6 and -1beta, and tumor necrosis factor-alpha, showing similar anti-inflammatory effect to that of PEP-1-FK506BP. Furthermore, co-treatment with SF peptide and PEP-1- FK506BP exhibited more enhanced anti-inflammatory effects than the samples treated with SF peptides or PEP- 1-FK506BP alone, suggesting the possibility that SF peptide and PEP-1-FK506BP might interact with each other. Moreover, the transduction data demonstrated that SF peptide did not affect the transduction of PEP-1- FK506BP into HaCaT cells and mice skin, indicating that the improvement of anti-inflammatory effect of PEP-1- FK506BP was not caused by enhanced transduction of PEP-1-FK506BP. Thus, these results suggest the possibility that co-treatment with SF peptide and PEP-1-FK506BP may be exploited as a useful therapy for various inflammationrelated diseases.     

Levosulpiride, (S)-(-)-5-Aminosulfonyl-N-[(1-ethyl-2-pyrrolidinyl) methyl]-2-methoxybenzamide, enhances the transduction efficiency of PEP-1-ribosomal protein S3 in vitro and in vivo

Many proteins with poor transduction efficiency were reported to be delivered to cells by fusion with protein transduction domains (PTDs). In this study, we investigated the effect of levosulpiride on the transduction of PEP-1 ribosomal protein S3 (PEP-1-rpS3), and examined its influence on the stimulation of the therapeutic properties of PEP-1-rpS3. PEP-1-rpS3 transduction into HaCaT human keratinocytes and mouse skin was stimulated by levosulpiride in a manner that did not directly affect the cell viability. Following 12-O-tetradecanoylphorbol- 13-acetate (TPA)-induced inflammation in mice, levosulpiride alone was ineffective in reducing TPA-induced edema and in inhibiting the elevated productions of inflammatory mediators and cytokines, such as cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6 and -1β, and tumor necrosis factor- α. Anti-inflammatory activity by PEP-1-rpS3 + levosulpiride was significantly more potent than by PEP-1-rpS3 alone. These results suggest that levosulpiride may be useful for enhancing the therapeutic effect of PEP-1-rpS3 against various inflammatory diseases. [BMB reports 2011; 44(5): 329-334].     

Molecular and functional characterization of turkey interferon.

The turkey interferon (TkIFN) gene encodes a signal peptide and a mature protein of 30 and 162 amino acids, respectively. TkIFN mRNA expression was induced by reoviral double-stranded RNA in fibroblasts. The recombinant TkIFN protein possessed species-specific antiviral activity and in synergy with lipopolysaccharide (LPS) induced bone marrow macrophages to produce nitric oxide (NO). LPS or TkIFN alone did not induce bone marrow macrophages to produce significant amounts of NO, which showed that TkIFN provided one of the two signals necessary to induce NO production in turkey macrophages. Unlike the anti-inflammatory nature of mammalian alpha/beta IFNs, TkIFN augmented the LPS-induced expression of interleukin-8, a proinflammatory cytokine. This finding suggests a role for TkIFN in inflammatory conditions.     

Structures of Bombyx mori and Samia cynthia ricini silk fibroins studied with solid-state NMR

There are many kinds of silks spun by silkworms and spiders, which are suitable to study the structure-property relationship for molecular design of fibers with high strength and high elasticity. In this review, we mainly focus on the structural determination of two well-known silk fibroin proteins that are from the domesticated silkworm, Bombyx mori, and the wild silkworm, Samia cynthia ricini, respectively. The structures of B. mori silk fibroin before and after spinning were determined by using an appropriate model peptide, (AG)(15), with several solid-state NMR methods; (13)C two-dimensional spin-diffusion solid-state NMR and rotational echo double resonance (REDOR) NMR techniques along with the quantitative use of the conformation-dependent (13)C CP/MAS chemical shifts. The structure of S. c. ricini silk fibroin before spinning was also determined by using a model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, with the solid-state NMR methods. The transition from the structure of B. mori silk fibroin before spinning to the structure after spinning was studied with molecular dynamics calculation by taking into account several external forces applied to the silk fibroin in the silkworm.     

Suppression of TNF-alpha-induced MMP-9 expression by a cell-permeable superoxide dismutase in keratinocytes

Up-regulation of selected matrix metalloproteinases (MMPs) such as MMP-9 contributes to inflammatory processes during the development of various skin diseases, such as atopic dermatitis. In this study, we examined the effect of a cell-permeable superoxide dismutase (Tat-SOD) on TNF-α-induced MMP-9 expression in human keratinocyte cells (HaCaT). When Tat-SOD was added to the culture medium of HaCaT cells, it rapidly entered the cells in dose- and time-dependent manners. Tat-SOD decreased TNF-α-induced reactive oxygen species (ROS) generation. Tat-SOD also inhibited TNF-α-induced NF-κB DNA binding activity. Treatment of HaCaT cells with Tat-SOD significantly inhibited TNF-α-induced mRNA and protein expression of MMP-9, as measured by RT-PCR and Western blot analysis. In addition, Tat-SOD suppressed TNF-α-induced gelatinolytic activity of MMP-9. Taken together, our results indicate that Tat-SOD can suppress TNF-α-induced MMP-9 expression via ROS-NF-κB-dependent mechanisms in keratinocytes, and therefore can be used as an immunomodulatory agent against inflammatory skin diseases related to oxidative stress.     

活络效灵丹抗炎镇痛作用的实验研究

目的：研究活络效灵丹的抗炎、镇痛、消肿等药理作用,为该药的临床研究提供基础。方法：用二甲苯致小鼠耳肿胀法和角又莱胶致大鼠足肿胀法研究抗炎作用,用热板法和扭体法研究镇痛作用。结果：活络效灵丹能较好地抑制二甲苯引起的小鼠耳廓炎性肿胀和大鼠甲醛致足跖肿胀,能显著提高热板试验小鼠的痛阈,有效抑制冰醋酸引起的小鼠扭体反应次数。结论：活络效灵丹具有良好的抗炎、镇痛、消肿等作用。     

Topically applied vitamin E prevents massive cutaneous inflammatory and oxidative stress responses induced by double application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice

Vitamin E (alpha-tocopherol) is a promising chemopreventive and pharmacologically safe agent, which can be exploited or tested against skin cancer. It is an established antioxidant with an ability to ameliorate the UV-induced skin damage and chemically induced inflammation in lungs. However, there are some conflicting reports about its role as a modulator of chemically induced promotion. We evaluated its efficacy in preventing the inflammatory and oxidative stress responses in a double 12-O-tetradecanoylphorbol-13-acetate (TPA) application tumor skin promotion protocol. Double application of TPA was undertaken to produce massive inflammatory and oxidative stress responses. Topical TPA treatment adversely altered many of the marker responses of stage I skin tumor promotion. Vitamin E application 30 min prior to TPA treatment (10 nmol) inhibited induction of hydrogen peroxide, myeloperoxidase (MPO) activity, xanthine oxidase (XO) activity and lipid peroxidation (LPO). Vitamin E also positively modulated altered antioxidants of mouse skin. Histological examination also revealed marked improvement. These results confirm the efficacy of vitamin E against early inflammatory and oxidative stress responses, which are hallmark of tumor promotion and provide rational basis for chemopreventive action of vitamin E in skin cancer.     

Preparation, characterization and biocompatibility of electrospinning heparin-modified silk fibroin nanofibers

In this study, the electrospun silk fibroin nanofibrous scaffolds were modified with heparin by grafting after plasma treatment and blending electrospinning. Morphology, microstructure, chemical composition and grafting efficiency of the heparin-modified silk fibroin nanofibrous scaffolds were characterized to evaluate the effect of modification by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectrometer (XPS). The results showed that the heparin was successfully introduced to the silk fibroin nanofibrous scaffolds by both the two kinds of modification, and there was a hydrogen bonding between the silk fibroin and heparin. Moreover, the hydrophilicity, O-containing groups and negative charge density of the heparin-modified scaffolds were enhanced. In vitro coagulation time tests showed that the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) of the heparin-modified scaffolds were much higher than those of the pure silk fibroin scaffolds. L929 fibroblasts and EVCs spread and proliferated better on the heparin-modified scaffolds than on the pure silk fibroin scaffolds. Macrophages, neutrophils and lymphocytes were not observed in the heparin-modified scaffolds, which indicated that the modified scaffolds could induce minor inflammation in vivo. The results indicated that the electrospun heparin-modified silk fibroin nanofibrous scaffolds could be considered as ideal candidates for tissue engineering scaffolds.     

Anti-inflammatory agents from Sandoricum koetjape Merr.

The anti-inflammatory activity of the stem extracts of Sandoricum koetjape was investigated on topical administration using the TPA (tetradecanoylphorbol acetate)-induced mouse ear inflammation model. Bioassay-guided chromatographic fractionation of active fractions led to the isolation 3-oxo-12-oleanen-29-oic acid and katonic acid as the bioactive principles responsible for the anti-inflammatory acitivity. The percentage of inhibition exhibited by 3-oxo-12-oleanen-29-oic acid was almost equivalent to indomethacin.     

Paeonol attenuates airway inflammation and hyperresponsiveness in a murine model of ovalbumin-induced asthma

Paeonol, the main active component isolated from Moutan Cortex, possesses extensive pharmacological activities such as anti-inflammatory, anti-allergic, and immunoregulatory effects. In the present study, we examined the effects of paeonol on airway inflammation and hyperresponsiveness in a mouse model of allergic asthma. BALB/c mice sensitized and challenged with ovalbumin were administered paeonol intragastrically at a dose of 100?mg/kg daily. Paeonol significantly suppressed ovalbumin-induced airway hyperresponsiveness to acetylcholine chloride. Paeonol administration significantly inhibited the total inflammatory cell and eosinophil count in bronchoalveolar lavage fluid. Treatment with paeonol significantly enhanced IFN-γ levels and decreased interleukin-4 and interleukin-13 levels in bronchoalveolar lavage fluid and total immunoglobulin E levels in serum. Histological examination of lung tissue demonstrated that paeonol significantly attenuated allergen-induced lung eosinophilic inflammation and mucus-producing goblet cells in the airway. These data suggest that paeonol exhibits anti-inflammatory activity in allergic mice and may possess new therapeutic potential for the treatment of allergic bronchial asthma.     

带有丝素重链信号肽序列的家蚕丝胶蛋白启动子驱动DsRed的瞬时分泌表达

为了探讨家蚕Bombyx mori丝素蛋白重链信号肽序列在中部丝腺组织中是否具有功能活性,根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,构建了带有丝素重链基因fib-H信号肽的家蚕丝胶-1(ser-1)启动子(ser-HS),用ser-HS驱动DsRed基因构建了分泌型瞬时表达载体pSK-SerHS-DsRed-polyA。转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed。家蚕注射载体后,可在中部丝腺腔中检测到红色荧光,表明瞬时表达的DsRed已分泌到丝腺腔内。据此提出克隆的fib-H信号肽序列在家蚕中部丝腺组织中具有信号肽的功能。     

The effect of gallium nitrate on synoviocyte MMP activity

Gallium, a group IIIa metal salt, has been demonstrated to be an effective immunosuppressive agent. Gallium has also been shown to inhibit the production of inflammatory cytokines, such as IL-1beta, produced by macrophage-like cells in vitro. To further characterize the effects of gallium on the inflammatory process, we examined the effects of gallium nitrate on matrix metalloproteinase (MMP) activity utilizing the rabbit synoviocyte cell line HIG-82. HIG-82 cells were incubated with IL-1beta and TPA, with and without increasing concentrations of gallium nitrate. Conditioned medium was collected and assayed for MMP activity using a synthetic substrate and substrate gel zymography. IL-1beta and TPA alone induced MMP activity in HIG-82 cells. A dose-dependent inhibition of IL-1beta and TPA stimulated MMP activity by gallium nitrate at increasing concentrations was observed. This study demonstrates that gallium nitrate can inhibit the activity of MMPs and may be useful as a modulator of inflammation in arthritis.     

NMR analysis of the fibronectin cell-adhesive sequence, Arg-Gly-Asp, in a recombinant silk-like protein and a model peptide

It is well established that by introducing the cell-adhesive sequence Arg-Gly-Asp (RGD) from fibronectin into Bombyx mori silk fibroin by covalent coupling or bioengineering techniques, excellent biomaterials have been developed with the modified silk fibroin. However, there is no report about the structure and dynamics of the RGD moiety in the silk fibroin. To clarify the origin of such a high cell adhesion character and to design new recombinant silk protein with higher cell adhesion ability, it is necessary to characterize the structure and dynamics of the RGD moiety introduced into silk fibroin. In this study, the structure and dynamics of the RGD moiety in a recombinant silk-like protein, SLPF(10), consisting of the repeated silk fibroin sequence (AGSGAG)(3) and the sequence ASTGRGDSPA including the RGD moiety, were studied using solution NMR. The (1)H, (15)N, and (13)C chemical shifts indicate that the RGD moiety, as well as the silk fibroin sequence, takes a random coil form with high mobility in aqueous solution. Next, a (13)C solid-state NMR study was performed on a (13)C selectively labeled model peptide, AGSGAG[3-(13)C]A(7)GSGAGAGSGGT[2-(13)C]G(19)R[1-(13)C]G(21)DSPAGGGAGAGSGAG. After formic acid treatment, an increase in the β-sheet fraction for the AGSGAG sequence and peak narrowing of the residues around the RGD moiety were observed in the dry state. The latter indicates a decrease in the chemical shift distribution although the RGD moiety is still in random coil. A decrease in the peak intensities of the RGD moiety in the swollen state after immersing it in distilled water was observed, indicating high mobility of the RGD sequence in the peptide in the swollen state. Thus, the random coil state of the RGD moiety in the recombinant silk-like protein is maintained in aqueous solution and also in both dry and swollen state. This is similar to the case of the RGD moiety in fibronectin. The presence of the linker ASTG at the N-terminus and SPAGG at the C-terminus seems important to maintain the random coil form and the flexible state of the RGD sequence in order to permit access for binding to various integrins.     

Anti-inflammatory activity of amylin and CGRP in different experimental models of inflammation

The anti-inflammatory activity of amylin was studied in different models of inflammation, and compared to that of CGRP. Both peptides were active against mouse ear oedema induced by croton oil and acetic acid-induced peritonitis in the rat. CGRP was more potent than amylin in both models. Pretreatment with CGRP 8–37 fragment blocked the anti-inflammatory activity of both peptides in croton oil ear oedema. No anti-inflammatory activity was evidenced against serotonin-induced rat paw oedema and plasma protein extravasation induced by dextran in rat skin. Our results suggest that amylin exerts anti-inflammatory activity only in inflammatory models characterized by a vascular component. This effect appears to be mediated by the involvement of CGRP receptors.     

Reduction of oxidative stress and cytokine-induced neutrophil chemoattractant (CINC) expression by red wine polyphenols in zinc deficiency induced intestinal damage of rat

Consumption of red wine has been associated with health promotion and disease prevention. We have previously found that the intestine of zinc-deficient (ZD) rats develop oxidative damage associated with inflammation. Here we have used this model to investigate whether red wine polyphenols could protect against intestinal injury and, if so, whether this protection was achieved through antioxidant and anti-inflammatory activity. The intestinal alterations induced by zinc deficiency such as morphological damage, increased TBA-RS level and CuZn-superoxide dismutase activity, and decreased glutathione peroxidase activity, did not develop with the administration to ZD rats of a suspension of dealcoholated red wine (RWS). The same treatment induced in control rats a decrease of TBA-RS level but also of glutathione peroxidase and catalase activity. Treatment with RWS to ZD rats prevented a marked mucosal macrophage and neutrophil infiltration. The expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha and cytokine-induced neutrophil chemoattractant (CINC), was induced by zinc deficiency, whereas that of the anti-inflammatory interleukin-10 was suppressed. Treatment with RWS reduced CINC expression. These results report a novel activity of red wine polyphenols in downregulation of intestinal CINC expression, which likely protects cells against inflammatory processes.     

Potentiated Interaction between Ineffective Doses of Budesonide and Formoterol to Control the Inhaled Cadmium-Induced Up-Regulation of Metalloproteinases and Acute Pulmonary Inflammation in Rats

The anti-inflammatory properties of glucocorticoids are well known but their protective effects exerted with a low potency against heavy metals-induced pulmonary inflammation remain unclear. In this study, a model of acute pulmonary inflammation induced by a single inhalation of cadmium in male Sprague-Dawley rats was used to investigate whether formoterol can improve the anti-inflammatory effects of budesonide. The cadmium-related inflammatory responses, including matrix metalloproteinase-9 (MMP-9) activity, were evaluated. Compared to the values obtained in rats exposed to cadmium, pretreatment of inhaled budesonide (0.5 mg/15 ml) elicited a significant decrease in total cell and neutrophil counts in bronchoalveolar lavage fluid (BALF) associated with a significant reduction of MMP-9 activity which was highly correlated with the number of inflammatory cells in BALF. Additionally, cadmium-induced lung injuries characterized by inflammatory cell infiltration within alveoli and the interstitium were attenuated by the pre-treatment of budesonide. Though the low concentration of budesonide (0.25 mg/15 ml) exerted a very limited inhibitory effects in the present rat model, its combination with an inefficient concentration of formoterol (0.5 mg/30 ml) showed an enhanced inhibitory effect on neutrophil and total cell counts as well as on the histological lung injuries associated with a potentiation of inhibition on the MMP-9 activity. In conclusion, high concentration of budesonide alone could partially protect the lungs against cadmium exposure induced-acute neutrophilic pulmonary inflammation via the inhibition of MMP-9 activity. The combination with formoterol could enhance the protective effects of both drugs, suggesting a new therapeutic strategy for the treatment of heavy metals-induced lung diseases.     

小茴香挥发油的抗炎镇痛作用

目的：研究小茴香挥发油的抗炎、镇痛作用,为指导临床合理用药提供科学依据。方法：应用二甲苯致小鼠耳廓肿胀、蛋清致大鼠足肿胀2种动物模型进行抗炎药效学实验;采用醋酸致小鼠扭体反应进行镇痛实验。结果：小茴香挥发油能显著抑制上述各种动物模型的炎症反应及醋酸引起的小鼠扭体反应。结论：小茴香挥发油具有抗炎和镇痛作用。