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Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1–4. Among these receptors, RXFP3 lacks a specific natural or synthetic agonist at present. A previously designed chimeric R3/I5 peptide, consisting of the B-chain of relaxin-3 and the A-chain of INSL5, displays equal activity towards the homologous RXFP3 and RXFP4. To increase its selectivity towards RXFP3, in the present study we conducted extensive mutagenesis around the B-chain C-terminal region of R3/I5. Decreasing or increasing the peptide length around the B23–B25 position dramatically lowered the activation potency of R3/I5 towards both RXFP3 and RXFP4. Substitution of B23Gly with Ala or Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained considerable activation potency towards RXFP4. Substitution of B24Gly increased the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. The best mutant, [G(B24)S]R3/I5, displayed 20-fold higher activation potency towards RXFP3 than towards RXFP4, meanwhile retained full activation potency at RXFP3. Thus, [G(B24)S]R3/I5 is the best RXFP3-selective agonist known to date. It is a valuable tool for investigating the physiological functions of RXFP3, and also a suitable template for developing RXFP3-specific agonists in future.

Insulin-like peptide 5 (INSL5) is a gut peptide hormone belonging to the insulin/relaxin superfamily. It is implicated in the regulation of food intake and glucose homeostasis by activating relaxin family peptide receptor 4 (RXFP4). Previous studies have suggested that the B-chain is important for INSL5 activity against RXFP4. However, functionalities of the B-chain residues have not yet been systematically studied. In the present work, we conducted alanine-scanning mutagenesis of the B-chain residues of human INSL5 to obtain an overview of their contributions. Binding and activation assays of these INSL5 mutants with human RXFP4 identified two essential exposed B-chain C-terminal residues (B23Arg and B24Trp) and one important exposed central B-chain residue (B16Ile). These three determinant residues together with the C-terminal carboxylate moiety probably constitute a central receptor-binding patch that forms critical hydrophobic and electrostatic interactions with RXFP4 during INSL5 binding. Some other exposed residues, including B10Glu, B12Ile, B13Arg, B17Tyr, B21Ser, and B22Ser, made minor contributions to INSL5 function. These auxiliary residues are scattered around the edge of the central receptor-binding patch, and thus form a peripheral receptor-binding patch on the surface of INSL5. Our present work provides new insights into the interaction mechanism of INSL5 with its receptor RXFP4.

The complete mitogenome sequence of the taiga shrew (Sorex isodon) was determined using long PCR. The genome was 17,008 bp in length and contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one origin of L strand replication and one control region. The overall base composition of the heavy strand is A (32.5%), C (24.5%), T (28.5%), and G (13.5%). The base compositions present clearly the A–T skew, which is most obviously in the control region and protein-coding genes. The extended termination-associated sequence domain, the central conserved domain and the conserved sequence block domain are defined in the mitochondrial genome control region of the taiga shrew. Mitochondrial genome analyses based on MP, ML, NJ, and Bayesian analyses yielded identical phylogenetic trees. The eight Sorex species formed a monophyletic group with the high bootstrap value (100%) in all examinations.

The complete mitogenome sequence of the slender shrew (Sorex gracillimus) was determined using long PCR. The genome was 17,002 bp in length and contained 13 protein-coding genes, two ribosomal RNA ...

There is a continuous need to improve the viral safety of plasma products, and we here report the development and optimization of a manufacturing-scale virus removal nanofiltration step for intravenous immunoglobulin (IVIG) using the recently introduced Planova™ BioEX filter. IVIG throughput was examined for various operating parameters: transmembrane pressure, temperature, protein concentration, and prefiltration methods. The developed procedure was based on filtering undiluted process solution (50.0 g/l IVIG) under constant transmembrane pressure filtration at 294 kPa and 25 °C following prefiltration with a 0.1 μm MILLEX VV filter. The recovery of IgG was approximately 98%, and no substantial changes in biochemical characteristics were observed before and after nanofiltration in scaled-up production. A viral clearance validation study with parvovirus under worst-case conditions performed at the National Institutes for Food and Drug Control of China (NIFDC) showed PPV logarithmic reduction value (LRV) > 4. Improved viral safety of IVIG can be assured by implementing a Planova BioEX nanofiltration step to ensure effective parvovirus clearance under conditions providing excellent protein recovery and no detectable impact on product biochemical properties. This plasma-derived IVIG product is the first to be certified for parvovirus safety by the NIFDC in China.

Ethnopharmacological relevance Pogostemon cablin (Blanco) Benth is a well-known medicinal herb commonly used in many Asian countries for inflammatory diseases. Pogostone (PO), a natural product isolated from Pogostemon cablin , is known to exert various pharmacological activities. This study aimed to investigate the anti-inflammatory property of PO, to elucidate its mechanism of action, and to evaluate its potential acute toxicity. Materials and methods The in vitro anti-inflammatory activity of PO was assessed using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The protein and mRNA levels of proinflammatory mediators were measured with ELISA and RT-PCR, respectively. Proteins of the NF- κ B and MAPK family were determined by Western blot to investigate the underlying molecular mechanisms. The in vivo anti-inflammatory activity of PO was tested using LPS-induced endotoxic shock in mice. In addition, the median lethal dose (LD 50 ) of PO in mice was tested in an acute toxicity test. Results In vitro, PO significantly inhibited the protein and mRNA expression of proinflammatory mediators including TNF-α, IL-6, IL-1β, NO, and PGE 2 . The action mechanism of the anti-inflammatory activity of PO was partly dependent on inhibition of the activation of NF- κ B and the phosphorylation of JNK and p38 MAPK. In vivo , PO was able to significantly reduce the mortality induced by LPS in mice. Furthermore, PO could markedly suppress the production of the proinflammatory mediators in serum, and attenuate liver and lung injury. The action mechanisms of PO during endotoxic shock may be attributed to down-regulation of the mRNA expression of inflammatory mediators in multiple organs via inhibition of the activation of NF- κ B and the phosphorylation of p38 MAPK. Moreover, the LD 50 of PO in mice was about 163 mg/kg with intravenous administration, which was about 8-fold higher than the dose used in the animal experiment. Conclusions Our findings regarding the anti-inflammatory effect of PO and the underlying molecular mechanisms help justify the use of Pogostemon cablin in Chinese medicine for the treatment of inflammatory diseases. More importantly, the results also render PO a promising anti-inflammatory agent worthy of further development into a pharmaceutical drug for the treatment of septic shock.

Bushen‑Yizhi formula (BSYZ), a traditional Chinese medicine formula consisting of six herbs has been reported to possess a neuroprotective effect. The present study aimed to investigate the effects of BSYZ on learning and memory abilities, as well as oxidative stress and neuronal apoptosis in the hippocampus of scopolamine (SCOP)‑induced senescence in mice, in order to reveal whether BSYZ is a potential therapeutic agent for Alzheimer's disease (AD). A high‑performance liquid chromatography (HPLC) fingerprint was applied to provide a chemical profile of BSYZ. Extracts of BSYZ were orally administered to mice with SCOP‑induced memory impairment for two weeks. The learning and memory abilities were determined by the Morris water maze test. The oxidant stress‑related indices, such as activity of superoxide dismutase (SOD) and levels of glutathione (GSH) and malondialdehyde (MDA) were examined in hippocampus of SCOP‑treated mice. The cell death ratio was assessed by TUNEL staining, while apoptotic‑related proteins including Bcl‑2 and Bax were determined by immuno-fluorescent staining and western blot analysis. Caspase‑3 was determined by western blot analysis. Consequently, a chromatographic condition, which was conducted at 35˚C with a flow rate of 0.8 ml/min on the Gemini C18 column with mobile phase of acetonitrile and water‑phosphoric acid (100:0.1, v/v), was established to yield common fingerprint chromatography under 203 nm with a similarity index of 0.986 within 10 batches of BSYZ samples. BSYZ at a dose of 2.92 g/kg significantly improved the cognitive ability, restored the abnormal activity of SOD and increased the levels of MDA and GSH induced by SCOP. Moreover, the neural apoptosis in the hippocampus of SCOP‑treated mice was reversed by BSYZ by regulating the expression of Bcl‑2, Bax and caspase‑3. The results demonstrated that BSYZ had neuroprotective effects in SCOP‑induced senescence in mice by ameliorating oxidative stress and neuronal apoptosis in the brain, supporting its potential in AD treatment.

In the present study, the inactivation effect of scutellarin (SL) on jack bean urease was investigated to elucidate the inhibitory potency, kinetics and mechanism of inhibition. It was revealed that SL acted as a concentration- and time-dependent inactivator of urease characteristic of slow-binding inhibition with an IC50 of 1.35±0.15 mM. The rapid formation of the initial SL-urease complex with an inhibition constant of Ki=5.37×10(-2) mM was followed by a slow isomerization into the final complex with the overall inhibition constant of Ki*=3.49×10(-3) mM. High effectiveness of thiol protectors, such as L-cysteine (L-cys), 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) significantly slowed down the rate of inactivation, indicating the strategic role of the active site sulfhydryl group in the blocking process. While the insignificant protection by boric acid and fluoride from the inactivation further confirmed that the active site cysteine should be obligatory for urease inhibition, which was also rationalized by the molecular docking study. The inhibition of SL on urease proved to be reversible since SL-blocked urease could be reactivated by DTT application and multidilution. The results obtained indicated that urease inactivation resulted from the reaction between SL and the sulfhydryl group.

Pogostemon cablin (Blanco) Benth. (Lamiaceae), which is named ‘Guang-Huo-Xiang’ in Chinese, has various therapeutic functions to remove dampness, relieve summer-heat and exterior syndrome, stop vomiting, and stimulate appetite. Patchouli oil is the essential oil of Pogostemonis herba. This study is aimed to investigate the antinociceptive and anti-allergy activities of patchouli oil. The antinociceptive activity of patchouli oil (20, 40, and 80 mg/kg) was evaluated using the acetic acid-induced writhing test (NIH mice) and the hot-plate test (NIH mice), and those for anti-allergy activity were tested using Schultz–Dale reaction, the passive cutaneous anaphylaxis (PCA) test, and delayed-type hypersensitivity (DTH) test. All doses of patchouli oil could significantly (p

The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1–4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4 and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous RXFP4 and RXFP3 remains unknown. In the present work, we chemically evolved INSL5 in vitro to a strong agonist of both RXFP4 and RXFP3 through replacement of its five B-chain residues with the corresponding residues of relaxin-3. We identified four determinants (B2Glu, B9Leu, B17Tyr and a rigid B-chain C-terminus) on INSL5 that are responsible for its inactivity at RXFP3. In reverse experiments, we grafted these determinants onto a chimeric R3/I5 peptide, which contains the B-chain of relaxin-3 and the A-chain of INSL5 and retains full activation potency at RXFP3 and RXFP4. All resultant R3/I5 mutants retained high activation potency towards RXFP4, but most displayed significantly decreased or even abolished activation potency towards RXFP3, confirming the role of these four INSL5 determinants in distinguishing RXFP4 from RXFP3.

Jacket is an ideal supporting structure for large 4 to 6 MW offshore wind turbine (OWT) in deep sea. However, Due to the harsh marine environment, as well as offshore wind farms are mostly located near a busy waterway, it is inevitable that ships crash offshore wind power structures. Sometimes, collision accidents may result in severe damage in the support structure. To study the collision behavior of jacket offshore structure, dynamic characteristics of the jacket OWT foundation is analyzed in this paper, based on ADINA explicit dynamic analysis method, considering different ship speed, impact locations and joint thickness. Based on the numerical results, the joints are reinforced in special location to meet requirements. Structural performances of jacket type OWT foundation under ship impact are studied. The research results in the paper can provide the reference for practical engineering design.Copyright © 2014 by ASME

Baicalin (BA) is the principal component of Radix Scutellariae responsible for its pharmacological activity. In this study, kinetics and mechanism of inhibition by BA against jack-bean urease were investigated for its therapeutic potential. It was revealed that the IC50of BA against jack-bean urease was2.74 ± 0.51 mM, which was proved to be a competitive and concentration-dependent inhibition with slow-binding progress curves. The rapid formation of initial BA-urease complex with an inhibition constant ofKi=3.89 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant ofKi*=1.47×10-4 mM. High effectiveness of thiol protectors against BA inhibition indicated that the strategic role of the active-site sulfhydryl group of the urease was involved in the blocking process. Moreover, the inhibition of BA was proved to be reversible due to the fact that urease could be reactivated by dithiothreitol but not reactant dilution. Molecular docking assay suggested that BA made contacts with the important activating sulfhydryl group Cys-592 residues and restricted the mobility of the active-site flap. Taken together, it could be deduced that BA was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for treatments on urease-related diseases.

The present work was designed to evaluate the in vitro and in vivo anti-Candida activity of pogostone (PO), a natural product isolated from Pogostemon cablin (Blanco) Benth. PO showed potent in vitro activity against clinical Candida spp. isolates tested in this study. PO and the reference drug voriconazole (VRC) were equally effective against all the fluconazole-resistant Candida albicans strains, with MIC ranging from 3.1 μg/ml to 50 μg/ml. Besides, PO was fungicidal against all Candida isolates with MFC ranging from 50 μg/ml to 400 μg/ml. By contrast, VRC was fungistatic as it failed to elicit a fungicidal effect against the Candida spp. isolates at the highest tested concentration (400 μg/ml). Furthermore, oral and topical PO administration effectively reduced the fungal load in vagina of vulvovaginal candidiasis mouse models. Topical PO administration (1.0-4.0 mg/kg) demonstrated higher activity against the vulvovaginal candidiasis than VRC (4.0 mg/kg). The pharmacokinetics and safety profile of PO were also investigated. The pharmacokinetics assay revealed that PO was easily absorbed after oral administration in mice, which might account for its in vivo anti-Candida effect. The acute toxicity test showed that the median lethal dose of PO in mice was 355 mg/kg, which was much higher than the daily dose used for the therapeutic experiments. This study demonstrated the potential of PO as a promising candidate for the treatment of Candida infections, particularly for vulvovaginal candidiasis.