Molecular hydrogen-induced salinity tolerance requires melatonin signalling in Arabidopsis thaliana.

Melatonin (MT) plays positive roles in salinity stress tolerance. However, the upstream signalling components that regulate MT are poorly understood. Here, we report that endogenous MT acts downstream of molecular hydrogen (H2 ) in the salinity response in Arabidopsis. The addition of hydrogen-rich water and expression of the hydrogenase1 gene (CrHYD1) from Chlamydomonas reinhardtii increased endogenous H2 and MT levels and enhanced salinity tolerance. These results were not observed in the absence of serotonin N-acetyltransferase gene (SNAT). H2 increased the levels of SNAT transcripts in the wild-type and CrHYD1 lines, which had lower Na+ /K+ ratios and higher levels of ion transport-related gene transcripts. These changes were not observed in atsnat/CrHYD1-4 hybrids. The increased MT-dependent Na+ extrusion observed in the CrHYD1 plants resulted, at least in part, from enhanced Na+ /H+ antiport across the plasma membrane. The endogenous H2 -induced MT-dependent regulation of ion and redox homeostasis was impaired in the atsnat/CrHYD1-4 hybrids. Taken together, these results demonstrate that MT-induced salinity tolerance is induced by a H2 signalling cascade that regulates ion and redox homeostasis in response to salinity.

Methyl-coenzyme M reductase-dependent endogenous methane enhances plant tolerance against abiotic stress and alters ABA sensitivity in Arabidopsis thaliana.

KEY MESSAGE: Our study firstly elaborated the underlying mechanism of endogenous CH4-induced abiotic tolerance, along with an alteration of ABA sensitivity by mimicking the endogenous CH4 production in MtMCR transgenic Arabidopsis. Endogenous methane (CH4) production and/or emission have been ubiquitously observed in stressed plants. However, their physiological roles remain unclear. Here, the methyl-coenzyme M reductase gene from Methanobacterium thermoautotrophicum (MtMCR), encoding the enzyme of methanogenesis, was expressed in Arabidopsis thaliana, to mimic the production of endogenous CH4. In response to salinity and osmotic stress, MtMCR expression was up-regulated in transgenic plants, resulting in significant increase of endogenous CH4 levels. Similar results were observed in abscisic acid (ABA) treatment. The functions of endogenous CH4 were characterized by the changes in plant phenotypes related to stress and ABA sensitivity during the germination and post-germination periods. When challenged with osmotic stress, a reduction in water loss and stomatal closure, were observed. Redox homeostasis was reestablished during osmotic and salinity stress, and ion imbalance was also restored in salinity conditions. The expression of several stress/ABA-responsive genes was up-regulated, and ABA sensitivity, in particularly, was significantly altered in the MtMCR transgenic plants. Together, our genetic study for the first time elaborated the possible mechanism of endogenous CH4-enhanced salinity and osmotic tolerance, along with an alteration of ABA sensitivity. These findings thus provided novel cues for understanding the possible roles of endogenous CH4 in plants.

Optimization of Collagenase Production by Pseudoalteromonas sp. SJN2 and Application of Collagenases in the Preparation of Antioxidative Hydrolysates.

Collagenases are the most important group of commercially-produced enzymes. However, even though biological resources are abundant in the sea, very few of these commercially popular enzymes are from marine sources, especially from marine bacteria. We optimized the production of marine collagenases by Pseudoalteromonas sp. SJN2 and investigated the antioxidant activities of the hydrolysates. Media components and culture conditions associated with marine collagenase production by Pseudoalteromonas sp. SJN2 were optimized by statistical methods, namely Plackett-Burman design and response surface methodology (RSM). Furthermore, the marine collagenases produced by Pseudoalteromonas sp. SJN2 were seen to efficiently hydrolyze marine collagens extracted from fish by-products, and remarkable antioxidant capacities of the enzymatic hydrolysates were shown by DPPH radical scavenging and oxygen radical absorbance capacity (ORAC) tests. The final optimized fermentation conditions were as follows: soybean powder, 34.23 g·L-1; culture time, 3.72 d; and temperature, 17.32 °C. Under the optimal fermentation conditions, the experimental collagenase yield obtained was 322.58 ± 9.61 U·mL-1, which was in agreement with the predicted yield of 306.68 U·mL-1. Collagen from Spanish mackerel bone, seabream scale and octopus flesh also showed higher DPPH radical scavenging rates and ORAC values after hydrolysis by the collagenase. This study may have implications for the development and use of marine collagenases. Moreover, seafood waste containing beneficial collagen could be used to produce antioxidant peptides by proteolysis.

Acid-Base Interactions of Polystyrene Sulfonic Acid in Amorphous Solid Dispersions Using a Combined UV/FTIR/XPS/ssNMR Study.

This study investigates the potential drug-excipient interactions of polystyrene sulfonic acid (PSSA) and two weakly basic anticancer drugs, lapatinib (LB) and gefitinib (GB), in amorphous solid dispersions. Based on the strong acidity of the sulfonic acid functional group, PSSA was hypothesized to exhibit specific intermolecular acid-base interactions with both model basic drugs. Ultraviolet (UV) spectroscopy identified red shifts, which correlated well with the color change observed in lapatinib-PSSA solutions. Fourier transform infrared (FTIR) spectra suggest the protonation of the quinazoline nitrogen atom in both model compounds, which agrees well with data from the crystalline ditosylate salt of lapatinib. X-ray photoelectron spectroscopy (XPS) detected increases in binding energy of the basic nitrogen atoms in both lapatinib and gefitinib, strongly indicating protonation of these nitrogen atoms. (15)N solid-state NMR spectroscopy provided direct spectroscopic evidence for protonation of the quinazoline nitrogen atoms in both LB and GB, as well as the secondary amine nitrogen atom in LB and the tertiary amine nitrogen atom in GB. The observed chemical shifts in the LB-PSSA (15)N spectrum also agree very well with the lapatinib ditosylate salt where proton transfer is known. Additionally, the dissolution and physical stability behaviors of both amorphous solid dispersions were examined. PSSA was found to significantly improve the dissolution of LB and GB and effectively inhibit the crystallization of LB and GB under accelerated storage conditions due to the beneficial strong intermolecular acid-base interaction between the sulfonic acid groups and basic nitrogen centers.

A rapid UPLC-MS/MS method for the determination of oleanolic acid in rat plasma and liver tissue: application to plasma and liver pharmacokinetics.

A reliable high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for oleanolic acid (OA) determination in rat plasma and liver tissue using glycyrrhetic acid as the internal standard (IS). Plasma and liver homogenate samples were prepared using solid-phase extraction. Chromatographic separation was achieved on a C18 column using an isocratic mobile phase system. The detection was performed by multiple reaction monitoring mode via positive electrospray ionization interface. The calibration curves showed good linearity (R(2) > 0.9997) within the tested concentration ranges. The lower limit of quantification for plasma and liver tissue was ≤0.75 ng/mL. The intra- and inter-day precision and accuracy deviations were within ±15% in plasma and liver tissue. The mean extraction recoveries ranged from 80.8 to 87.0%. In addition, the carryover, matrix effect, stability and robustness involved in the method were also validated. The method was successfully applied to the plasma and hepatic pharmacokinetics of OA after oral administration to rats.

Investigation of drug-excipient interactions in lapatinib amorphous solid dispersions using solid-state NMR spectroscopy.

This study investigated the presence of specific drug-excipient interactions in amorphous solid dispersions of lapatinib (LB) and four commonly used pharmaceutical polymers, including Soluplus, polyvinylpyrrolidone vinyl acetate (PVPVA), hydroxypropylmethylcellulose acetate succinate (HPMCAS), and hydroxypropylmethylcellulose phthalate (HPMCP). Based on predicted pKa differences, LB was hypothesized to exhibit a specific ionic interaction with HPMCP, and possibly with HPMCAS, while Soluplus and PVPVA were studied as controls without ionizable functionality. Thermal studies showed a single glass transition (Tg) for each dispersion, in close agreement with predicted values for Soluplus, PVPVA, and HPMCAS systems. However, the Tg values of LB-HPMCP solid dispersions were markedly higher than predicted values, indicating a strong intermolecular interaction between LB and HPMCP. (15)N solid-state NMR provided direct spectroscopic evidence for protonation of LB (i.e., salt formation) within the HPMCP solid dispersions. (1)H T1 and (1)H T1ρ relaxation studies of the dispersions supported the ionic interaction hypothesis, and indicated multiple phases in the cases of excess drug or polymer. In addition, the dissolution and stability behavior of each system was examined. Both acidic polymers, HPMCAS and HPMCP, effectively inhibited the crystallization of LB on accelerated stability, likely owing to beneficial strong intermolecular hydrogen and/or specific ionic bonds with the acidic polymers. Soluplus and PVPVA showed poor physical properties on stability and subsequently poor crystallization inhibition.

Hydrophilic modification of titania nanomaterials as a biofunctional adsorbent for selective enrichment of phosphopeptides.

TiO2-based metal oxide affinity chromatography (MOAC) nanomaterials show high potential in phosphoproteome mass-spectrometric (MS) analysis. However, a drawback of TiO2 nanomaterials is poor water solubility, which greatly reduces the enrichment efficiency of phosphopeptides and eventually limits their use in phosphoproteome MS analysis. In this work, a hydrophilic TiO2 hybrid material (denoted as NH2@TiO2) is successfully designed with 1,6-hexanediamine modified on the surface of TiO2 nanoparticles and applied as a biofunctional adsorbent for selective enrichment of phosphopeptides. The novel TiO2 hybrid material with high hydrophilicity and biocompatibility is characterized using scanning electron microscopy (SEM), energy dispersive X-ray (EDX) and infrared (IR) spectroscopy, and its performance in selective enrichment of phosphopeptides is evaluated with the standard protein digests, human serum and the tryptic digests of nonfat milk.

Effects of preparing techniques and aging on dissolution behavior of the solid dispersions of NF/Soluplus/Kollidon SR: identification and classification by a combined analysis by FT-IR spectroscopy and computational approaches.

CONTEXT: Pharmaceutical solid dispersions are known to be seriously affected by issues of aging and processing. OBJECTIVE: This study investigated the spectral patterns in the solid dispersions (SD) of Nifedipine/Soluplus/Kollidon SR and the feasibility of the methodology in identification and evaluation of the solid dispersions. METHODS: The SD samples were prepared by hot melt extrusion (HMESD), solvent-evaporation (SESD), and fusion-cooling (FCSD). In order to distinguish the different SD samples, a combined analytical strategy by FT-IR spectrum, Raman spectrum, and computational approaches (PCA and HCA) were developed to investigate the spectral patterns of the solid dispersions. Differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), scanning electron microscope (SEM), and dissolution test were employed as the reference characterization. The stability test under the accelerated condition was carried out to investigate the physical stability of the SDs. RESULT: For the three prepared SDs, the evident differences on the dissolution behaviors and the trend of aging was observed. By means of the combined analytical strategy, the samples could be successfully identified in terms of their preparing techniques. The strength of hydrogen bonding interaction between NF and polymers decreased in the order of HMESD > SESD > FCSD. The results of the stability test indicated that the similarity factor f2 value of dissolution profile decreased in the order of HMESD > SESD > FCSD. HMESD exhibited a tendency of minimal changing on both dissolution behavior and spectral patterns. CONCLUSION: The combined strategy suggested the possibility for identification of specific SDs in quality control and prediction of their trends on the aging.

Influence of different polymers on crystallization tendency and dissolution behavior of cilnidipine in solid dispersions.

CONTEXT: Cilnidipine (CN) is a novel dihydropyridine calcium antagonist that is practically insoluble in aqueous media and exhibits a low oral bioavailability or limited clinical efficacy. OBJECTIVE: This study investigated the effects of three commercial and chemically diverse polymers - PVP, PVP/VA and Soluplus - on crystallization tendency and in vitro dissolution profiles of CN in order to determine an optimum carrier for composing the preferred solid dispersion (SD) of CN. METHODS: All these co-evaporated systems were characterized up to 3 months by thermoanalytical (DSC), crystallographic (POM, PXRD), microscopic (SEM) and spectroscopic (FTIR) techniques. RESULTS: The results showed that the polymers could be sorted by their effects of inhibiting CN crystallization in the ascending order: Soluplus, PVP/VA, PVP. The sequence was in accordance with that of the strength of drug-polymer hydrogen bonds revealed by FTIR spectra. It could be ascribed to relative hydrogen-bonding acceptor strengths of N-vinylpyrrolidone moiety in the polymer molecules. On the other hand, all the SDs showed enhanced dissolution profiles compared to pure CN alone. On their effects of enhancing CN dissolution, the polymers could be sorted in the descending order: Soluplus, PVP, PVP/VA. CONCLUSIONS: It implied that the dissolution behavior of CN could bear a close relationship to both hydration capacity and hydrogen-bonding interaction tendency of moieties of the polymers. It might suggest an optimal formulation for CN comprising both PVP and Soluplus.

Simultaneous quantification of paeoniflorin, nobiletin, tangeretin, liquiritigenin, isoliquiritigenin, liquiritin and formononetin from Si-Ni-San extract in rat plasma and tissues by liquid chromatography-tandem mass spectrometry.

In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of seven bioactive components including paeoniflorin, nobiletin, tangeretin, liquiritigenin, isoliquiritigenin, liquiritin and formononetin in rat plasma and tissues after oral administration of Si-Ni-San extract using astragaloside IV as internal standard (IS). The plasma and tissue samples were extracted by solid-phase extraction. Chromatographic separation was accomplished on a C18 column with a multiple-step gradient elution. The quantification was obtained by scanning with multiple reaction monitoring via an electrospray ionization source that was operated by switching between the positive and negative modes in two MS/MS scan segments. Full validation of the assay was implemented. In conclusion, this method demonstrated good linearity and specificity. The lower limits of quantification for the analytes were <7.5 ng/mL. Intra- and inter-day precisions (RSD) were <12.5% and accuracy (RE) ranged from -10.2 to 7.3%. The average recoveries of the analytes from rat plasma and tissues were >65.2% and 58.6%, respectively. The validated method was further applied to the determination of actual rat plasma and tissues after oral administration of Si-Ni-San extract. The results provided a meaningful basis for the clinical application of this prescription.

Rapid screening and quantification of sulfonate derivatives in white peony root by UHPLC-MS-MS.

A rapid ultra-high-performance liquid chromatographic-tandem mass spectrometric (UHPLC-MS-MS) method has been developed for rapid screening and quantitative analysis of sulfonate derivatives (SDs) in commercial white peony root. Separation was performed on an Agilent Zorbax Eclipse Plus-C18 column by gradient elution with acetonitrile-0.1% (v/v) formic acid as the mobile phase. In-source fragmentation was used to generate the characteristic fragment ion at m/z 259 and to screen for nine SDs. Detection of these SDs was further performed in multiple reaction monitoring (MRM) mode to improve sensitivity and to quantify the two SDs paeoniflorin sulfonate and benzoylpaeoniflorin sulfonate. The method was validated for specificity, linearity, limits of detection and quantification, precision, accuracy, and matrix effects. Nine commercial white peony root samples were examined by use of this method, which revealed great variety in the paeoniflorin sulfonate and benzoylpaeoniflorin sulfonate content.

Methane control of cadmium tolerance in alfalfa roots requires hydrogen sulfide.

Hydrogen sulfide (H2S) is well known as a gaseous signal in response to heavy metal stress, while methane (CH4), the most prevalent greenhouse gas, confers cadmium (Cd) tolerance. In this report, the causal link between CH4 and H2S controlling Cd tolerance in alfalfa (Medicago sativa) plants was assessed. Our results observed that the administration of CH4 not only intensifies H2S metabolism, but also attenuates Cd-triggered growth inhibition in alfalfa seedlings, which were parallel to the alleviated roles in the redox imbalance and cell death in root tissues. Above results were not observed in roots after the removal of endogenous H2S, either in the presence of either hypotaurine (HT; a H2S scavenger) or DL-propargylglycine (PAG; a H2S biosynthesis inhibitor). Using in situ noninvasive microtest technology (NMT) and inductively coupled plasma mass spectroscopy (ICP-MS), subsequent results confirmed the participation of H2S in CH4-inhibited Cd influx and accumulation in roots, which could be explained by reestablishing glutathione (GSH) pool (reduced/oxidized GSH and homoglutathione) homeostasis and promoting antioxidant defence. Overall, our results clearly revealed that H2S operates downstream of CH4 enhancing tolerance against Cd stress, which are significant for both fundamental and applied plant biology.

Deciphering the synergistic network regulation of active components from SiNiSan against irritable bowel syndrome via a comprehensive strategy: Combined effects of synephrine, paeoniflorin and naringin.

BACKGROUND: SiNiSan (SNS) is an ancient Chinese herbal prescription, and the current clinical treatment of irritable bowel syndrome (IBS) is effective. In the previous study of the research team, the multi-functional co-synergism of SNS against IBS was presented. Some potential drug targets and candidate ligands were predicted. PURPOSE: This study attempts to explore the crucial ingredient combinations from SNS formula and reveal their synergistic mechanism for IBS therapy. MATERIALS AND METHODS: In present study, a comprehensive strategy was performed to reveal IBS related pathways and biological modules, and explore synergistic effects of the ingredients, including ADME (absorption, distribution, metabolism, excretion) screening, Text mining, Venn analysis, Gene ontology (GO) analysis, Pathway cluster analysis, Molecular docking, Network construction and Experimental verification in visceral hypersensitivity (VHS) rats. RESULTS: Three compressed IBS signal pathways were derived from ClueGO KEGG analysis of 63 IBS genes, including Neuroactive ligand-receptor interaction, Inflammatory mediator regulation of TRP (transient receptor potential) channels and Serotonergic synapse. A multi-module network, composed of four IBS therapeutic modules (psychological, inflammation, neuroendocrine and cross-talk modules), was revealed by Target-Pathway network. Nine kernel targets were considered closely associated with the IBS pathways, including ADRA2A, HTR2A, F2RL1, F2RL3, TRPV1, PKC, PKA, IL-1Β and NGF. In silico analysis revealed that three crucial ingredients (synephrine, paeoniflorin and naringin) were assumed to coordinate the network of those IBS therapeutic modules by acting on these kernel targets in the important pathways. In vivo experimental results showed that the crucial ingredient combinations synergistically affected the expressions of the kernel biological molecules, and improved the minimum capacity threshold of AWR in VHS rats. CONCLUSION: The study proposes the important IBS associated pathways and the network regulation mechanisms of the crucial ingredients. It reveals the multi-target synergistic effect of the crucial ingredient combinations for the novel therapy on IBS.

Deciphering the multicomponent synergy mechanisms of SiNiSan prescription on irritable bowel syndrome using a bioinformatics/network topology based strategy.

BACKGROUND: SiNiSan (SNS) is a traditional Chinese medicine (TCM) prescription that has been widely used in the clinical treatment of irritable bowel syndrome (IBS). However, the underlying active substances and molecular mechanisms remain obscure. PURPOSE: A bioinformatics/topology based strategy was proposed for identification of the drug targets, therapeutic agents and molecular mechanisms of SiNiSan against irritable bowel syndrome. MATERIALS AND METHODS: In this work, a bioinformatics/network topology based strategy was employed by integrating ADME filtering, text mining, bioinformatics, network topology, Venn analysis and molecular docking to uncover systematically the multicomponent synergy mechanisms. In vivo experimental validation was executed in a Visceral Hypersensitivity (VHS) rat model. RESULTS: 76 protein targets and 109 active components of SNS were identified. Bioinformatics analysis revealed that 116 disease pathways associated with IBS therapy could be classified into the 19 statistically enriched functional sub-groups. The multi-functional co-synergism of SNS against IBS were predicted, including inflammatory reaction regulation, oxidative-stress depression regulation and hormone and immune regulation. The multi-component synergetic effects were also revealed on the herbal combination of SNS. The hub-bottleneck genes of the protein networks including PTGS2, CALM2, NOS2, SLC6A3 and MAOB, MAOA, CREB1 could become potential drug targets and Paeoniflorin, Naringin, Glycyrrhizic acid may be candidate agents. Experimental results showed that the potential mechanisms of SiNiSan treatment involved in the suppression of activation of Dopaminergic synapse and Amphetamine addiction signaling pathways, which are congruent with the prediction by the systematic approach. CONCLUSION: The integrative investigation based on bioinformatics/network topology strategy may elaborate the multicomponent synergy mechanisms of SNS against IBS and provide the way out to develop new combination medicines for IBS.

Exploring the multicomponent synergy mechanism of Banxia Xiexin Decoction on irritable bowel syndrome by a systems pharmacology strategy.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia Xiexin Decoction (BXD) is a representative prescription to regulate spleen and stomach in "Treatise on Febrile Diseases", which has been proven effective for the clinical treatment of irritable bowel syndrome (IBS) in the past decades. However, the active principles and molecular mechanisms involved in BXD against IBS are vague yet. AIM OF THE STUDY: To unfold multicomponent synergy mechanism of BXD on irritable bowel syndrome, this work explored active principles, drug targets and crucial pathways using a systems pharmacology strategy. MATERIALS AND METHODS: In this study, a systems pharmacology based strategy was applied by the procedures integrating compound database construction, ADME evaluation, target identification, functional annotation, pathway enrichment analysis, network analysis, and molecular docking verification. The 158 compounds from BXD were selected for the screening. The Compound-Target network (C-T) and the Target-Pathway network (T-P) were constructed. The bioinformatics and network topology were employed to systematically reveal multicomponent-target interactions of BXD. The affinity between important ingredients and the kernel targets was validated using molecular mechanics simulation. RESULTS: The 35 potential important ingredients and the 16 associated kernel targets were identified. 27 crucial pathways, in which the kernel targets participated, could regulate the biological processes, such as synthesis of inflammatory mediators, smooth muscle relaxation and synaptic plasticity, closely related to pathological mechanism of IBS. The cross-talk interactions were revealed between TNF signaling pathway, Dopaminergic synapse and cGMP-PKG signaling pathway, which would exert the synergistic influences on the occurrence and treatment of the IBS. PTGS2, CALM, NOS2, SCN5A, and PRSS1 might become novel drug targets for IBS. CONCLUSIONS: The study demonstrated that the synergy molecular mechanisms of BXD mainly involved three therapeutic modules including inhibiting inflammatory reaction, maintaining intestinal function and improving psychological regulation via the multicomponent-target interaction networks. It may also provide the promising drug targets and therapeutic agents for the development of new medicines.

Effect of HPbetaCD on solubility and transdermal delivery of capsaicin through rat skin.

We evaluated the ability of hydroxypropyl-beta-cyclodextrin (HPbetaCD) to influence the percutaneous absorption of capsaicin (CP) through isolated rat skin. Phase solubility analysis and phase distribution studies suggested the potential of HPbetaCD as a solubilizer and permeation enhancer for CP. In vitro permeation studies showed the trend that, the penetration flux (J(s)) of CP increased with the increasing concentration of HPbetaCD from 0 to 2.20% (w/v), and then decreased dramatically when the concentration of HPbetaCD kept on increasing up to 15% (w/v). 2.20% (w/v) of HPbetaCD provided both just adequate solubilization and preferred J(s) for the permeation of CP (0.075%, w/v). Similar change patterns of the permeation parameters were also observed in the hydrogels, but the J(s) of CP was reduced significantly along with the increasing concentration of Carbopol U21. Histological analysis showed an invasive action of HPbetaCD on the stratum corneum (SC) of rat skin, which could only reduce the lag time (T(L)) but could not increase the J(s) of CP. On the other hand, the complexation of HPbetaCD with CP could attenuate this invasive action. It is inferred that excess of HPbetaCD could not only disturb the percutaneous absorption of CP but also disrupt the structure of SC.

Antioxidant and anti-freezing peptides from salmon collagen hydrolysate prepared by bacterial extracellular protease.

Extracted salmon skin collagen was hydrolysed with the free or immobilized extracellular protease of Vibrio sp. SQS2-3. The hydrolysate exhibited anti-freezing activity (>3 kDa) and antioxidant activity (<3000 Da) after ultrafiltration. The antioxidant peptide was further purified by size-exclusion chromatography and found to scavenge DPPH (73.29 ±â€¯1.03%), OH (72.73 ±â€¯3.34%,), and intracellular ROS in HUVECs; protect DNA against oxidation-induced damage; and have an ORAC of 2.78 ±â€¯0.28 mmol TE/g. The antioxidant peptide fraction was identified using mass spectrometry, and nineteen salmon collagen-sourced peptides were obtained. Of these, the peptide Pro-Met-Arg-Gly-Gly-Gly-Gly-Tyr-His-Tyr is a novel sequence and was the major component; this peptide was shown to have antioxidant activity via the ORAC assay (2.51 ±â€¯0.14 mmol TE/g). These results suggested that the protease from Vibrio sp. SQS2-3 is suitable for preparation of anti-freezing peptides and antioxidant peptides in a single step and represents a comprehensive use of fish skin collagen.

[Preparation and antioxidant activity detection of collagen peptide from Cirrhinus molitorella skin].

In order to prepare antioxidant peptide through hydrolyzing low-value protein resources with bacterial extracellular proteases and to discover novel proteases, crude extracellular protease from Pseudoalteromonas sp. SHK1-2 was obtained through fermentation which was used to hydrolyze collagen extracted from Cirrhinus molitorella skin. Small peptide fraction was isolated from hydrolysate by ultrafiltration and Sephadex LH-20 size exclusion chromatography and showed 1, 1-diphenyl-2-picrylhydrazyl radical scavenging activity (35.6%±7%), oxygen radical absorbance capacity and inhibition of DNA oxidation damage. The molecule weight was 776.2 Da, and amino acid sequence was Thr-Ala-Gly-His-Pro- Gly-Thr-His through liquid chromatography mass spectrum. Our findings suggest that peptide obtained from low-value protein of fish waste by hydrolysis with bacterial protease has antioxidant activity.

Improving Production of Protease from Pseudoalteromonas sp. CSN423 by Random Mutagenesis.

Pseudoalteromonas sp. CSN423, a marine strain, can express a major protease designated as E423 and it was secreted into the supernatant. To improve the protease E423 yield, Pseudoalteromonas sp. CSN423 was subjected to mutagenesis using UV irradiation. Mutant strain with 5.1-fold higher protease yield was isolated and named as Pseudoalteromonas sp. CSN423-M. Three protease bands were detected by zymography with casein as substrate, and results of mass spectrometry (MS) showed that two lower molecular weight protein bands were the same protease but with different mature forms. The entire protease operon was sequenced and no mutation was found. Mutant strain-associated changes of expression levels of protease synthesis and secretion-related genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Mutant strain had higher expression of e423 than wild-type strain. Such result was consistent with protease activity profiles. Moreover, the mutant strain had higher transcriptional levels of citrate synthase (cs), α-ketoglutarate decarboxylase (kgd), cytochrome c oxidase subunit I (coxI), tolC, hlyD (membrane protein), luxR3, luxO, and luxT (transcriptional regulator). However, hexokinase (hk), pyruvate dehydrogenase E1 (pd-e1), epsD (membrane protein), and luxR1 remained unchanged, and luxR2 decreased sharply in the mutant. These results suggested that the redox pathway was promoted in the mutant strain, and LuxR family transcriptional regulators in Pseudoalteromonas spp. may play some role in regulating protease expression. Meanwhile, the secretion of extracellular protease was closely related to ABC transport system. These results may shed some light on the molecular mechanism underlying higher yield of protease E423 from Pseudoalteromonas sp. CSN423-M.

In situ demonstration and characteristic analysis of the protease components from marine bacteria using substrate immersing zymography.

Zymography is a widely used technique for the study of proteolytic activities on the basis of protein substrate degradation. In this study, substrate immersing zymography was used in analyzing proteolysis of extracellular proteases. Instead of being added directly into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, the substrates were added into the immersing solution after electrophoresis. Substrate immersing zymography could accurately determine the molecular weight of trypsin, and band intensities were linearly related to the amount of protease. The diversity of extracellular proteases produced by different marine bacteria was analyzed by substrate immersing zymography, and large variations of proteolysis were evidenced. The proteolytic activity of Pseudoalteromonas strains was more complicated than that of other strains. Five Pseudoalteromonas strains and five Vibrio strains were further analyzed by substrate immersing zymography with different substrates (casein and gelatin), and multiple caseinolytic and gelatinolytic profiles were detected. The extracellular proteolytic profiles of Pseudoalteromonas strains exhibited a large intraspecific variation. Molecular weight (Mw) of the main protease secreted by Vibrio was 35 kDa. Additionally, the time-related change trends of the activities of extracellular proteases produced by Pseudoalteromonas sp. SJN2 were analyzed by substrate immersing zymography. These results implied the potential application of substrate immersing zymography for the analysis of the diversity of bacterial extracellular proteases.