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An alginate lyase producing bacterial strain, Cobetia sp. WG-007, was isolated and identified from rotting seaweed. The alginate lyase, Aly-W02, was purified by procedures of ultrafiltration, Q-Sepharose Fast Flow, Phenyl Sepharose 6 Fast Flow, and Superdex-G100 with specific activity of 21,285.5 U/mg. Aly-W02 had an apparent molecular mass of 35 kDa. It exhibited maximum activity at 45 °C in 50 mM sodium phosphate buffer (pH 8.5). This alginate lyase was stable in the pH range of 6.0-8.5. Among the tested metal ions, the addition of K

Two peptides of Pt-A (Glu-Asn-Trp 429 Da) and Pt-B (Glu-Gln-Trp 443 Da) were isolated from venom liquor ofDeinagkistrodon acutus. Their antiplatelet aggregation effects were evaluated with platelet-rich human plasmain vitro; the respective IC50of Pt-A and Pt-B was 66 μM and 203 μM. Both peptides exhibited protection effects on ADP-induced paralysis in mice. After ADP administration, the paralysis time of different concentration of Pt-A and Pt-B lasted as the following: 80 mg/kg Pt-B (152.8 ± 57.8 s) < 40 mg/kg Pt-A (163.5 ± 59.8 s) < 20 mg/kg Pt-A (253.5 ± 74.5 s) < 4 mg/kg clopidogrel (a positive control, 254.5 ± 41.97 s) < 40 mg/kg Pt-B (400.8 ± 35.9 s) < 10 mg/kg Pt-A (422.8 ± 55.4 s), all of which were statistically shorter than the saline treatment (666 ± 28 s). Pulmonary tissue biopsy confirmed that Pt-A and Pt-B prevented the formation of thrombi in the lung. Unlike ADP injection alone, which caused significant reduction of peripheral platelet count, Pt-A treatment prevented the drop of peripheral platelet counts; interestingly, Pt-B could not, even though the same amount of Pt-B also showed protection effects on ADP-induced paralysis and thrombosis. More importantly, intravenous injection of Pt-A and Pt-B did not significantly increase the hemorrhage risks as clopidogrel.

Background Studies on endophytes, a relatively under-explored group of microorganisms, are currently popular amongst biologists and natural product researchers. A fungal strain (ME4-2) was isolated from flower samples of mistletoe (Viscum coloratum) during a screening program for endophytes. As limited information on floral endophytes is available, the aim of the present study is to characterise fungal endophytes using their secondary metabolites. Results ME4-2 grew well in both natural and basic synthetic media but produced no conidia. Sequence analysis of its internal transcribed spacer rDNA demonstrated that ME4-2 forms a distinct branch within the genus Lasiodiplodia and is closely related to L. pseudotheobromae. This floral endophyte was thus identified as Lasiodiplodia sp. based on its molecular biological characteristics. Five aromatic compounds, including cyclo-(Trp-Ala), indole-3-carboxylic acid (ICA), indole-3-carbaldehyde, mellein and 2-phenylethanol, were found in the culture. The structures of these compounds were determined using spectroscopic methods combined with gas chromatography. To the best of our knowledge, our work is the first to report isolation of these aromatic metabolites from a floral endophyte. Interestingly, ICA, a major secondary metabolite produced by ME4-2, seemed to be biosynthesized via an unusual pathway. Furthermore, our results indicate that the fungus ME4-2 is a potent producer of 2-phenylethanol, which is a common component of floral essential oils. Conclusions This study introduces a fungal strain producing several important aromatic metabolites with pharmaceutical or food applications and suggests that endophytic fungi isolated from plant flowers are promising natural sources of aromatic compounds.

Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) resulting from chronic liver diseases. Efficient and well-tolerated drugs for its treatment are urgently needed. This study aims to identify the active ingredients of Antrodia camphorata by a bioassay-guided fractionation approach and explore the acting mechanism by using a hepatic stellate cell (HSC) line CFSC-8B stimulated by transforming growth factor-β1 (TGF-β1).The accumulation of collagens was evaluated using chromogenic precipitation reaction with picro-sirius red (PSR) dye solution and quantified by spectrophotometric analysis of the dissolved stain. MTT assay, cell migration assay, quantitative polymerase chain reaction and western blotting analysis were used to determine the cell viability, cell migration and gene expression.We established a rapid colorimetric assay suitable for screening of anti-hepatofibrotic reagents. Stimulation with 10 ng/mL TGF-β1 for 48 h and 200 μL PSR dye solution were optimal for the colorimetric assay in CFSC-8B cells. We used SB431542, silybin and another 11 antifibrotic reagents to verify the cellular model. Within the safe doses, they attenuated ECM production induced by TGF-β1. Bioactivity-guided fractionation led to the identification of antrodin B from A. camphorata. Antrodin B significantly ameliorated cell proliferation, cell migration, suppressed HSC activation marker α-smooth muscle actin expression and ECM components Col1, Col3 and Fn expression, and blocked the phosphorylation of Smad2/3 induced by TGF-β1 in CFSC-8B cells in a dose-dependent manner.We developed a simple assay based on TGF-β1-induced total collagen accumulation in CFSC-8B cells and identified antrodin B which may serve as a potential candidate for treatment of liver fibrosis.

Nitrilase-mediated biocatalysis has attracted substantial attention for its application in carboxylic acid production in recent years. In the present study, the fungus CA3-1 was isolated and identified as Gibberella intermedia based on its morphology, its 18S ribosomal DNA (rDNA), and internal transcribed spacer (ITS) sequences. The enzymatic properties of G. intermedia resting cells were determined, and the optimum activity was achieved at 40 °C with pH 7.6. The half-lives of the nitrilase at 30, 40, and 50 °C were 231.1, 72.9, and 6.4 h, respectively. This Gibberella nitrilase showed a wide substrate spectrum with high specificity for heterocyclic and aliphatic nitriles. It remained extremely active in 5% propanol. The presence of Ag(+), Hg(2+), and excess substrate inhibited the nitrilase activity, whereas Fe(2+), Mn(2+), and Li(+) improved enzyme activity. 3-Cyanopyridine (50 mM) was hydrolyzed into nicotinic acid within 30 min, whereas only5% of nicotinamide was detected. The results show that this fungal nitrilase is a promising candidate for commercial application in nicotinic acid production.

Inflammation is the responses of body to harmful stimuli in many pathological conditions. Prevention and treatment of inflammation are the main indication of a variety of natural products. Recently, Niuchangchih (Antrodia camphorata), a medicinal mushroom in Taiwan, has received considerable attention from the public due to its potent bioactivities, such as anti-inflammation, anti-cancer, immuno-modulatory and anti-hepatitis activities1. Till now, researchers have identified a total of 26 compounds from Niuchangchih, including 11 succinic/maleic acid derivatives, 8 triterpenoids, 1 benzenoids, 1 benzoquinone derivative, 5 miscellaneous compounds and polysaccharides, that possess anti-inflammatory effect2, 3. However, the mechanisms underlying their anti-inflammatory actions remain elusive. A recent study by Chen et al elucidated the molecular mechanisms of anti-inflammation of Niuchangchih. The authors isolated and purified 5 major antcins (A, B, C, H, and K) from fruiting bodies of Niuchangchih. They found that antcin A was most similar to glucocorticoids among the 5 compounds in the chemical structure. Furthermore, they demonstrated in human lung cancer cell A549 that antcin A was the active ingredient responsible for the anti-inflammatory effect of Niuchangchih, which might act via the same molecular mechanism triggered by glucocorticoids4. Finally, they showed the docking of antcin A to glucocorticoid receptor (GR) in a molecular modeling study. Thus, via mimicking glucocorticoids, antcin A may diffuse across the cell membrane and bind to the cytosolic GR that forms a dimmer after dissociated from the heat-shock protein (HSP) and then translocates into the nucleus to initiate the suppression of inflammation at the gene regulation level. Molecular docking showed that C-7 of antcin A was attached to the hydrophobic side of the steroidal backbone of GR, while C-7 of the other antcins was attached to the hydrophilic group, thus being expelled when docking to the binding cavity of GR. Chen's work is an important landmark in research of anti-inflammatory compounds in Niuchangchih. Hsien et al recently showed that the anti-inflammatory activity of antrocamphin A, another anti-inflammatory compound from Niuchangchih, resulted from suppressing pro-inflammatory molecule release via down-regulation of iNOS and COX-2 expression through NF-κB pathway5. Both the studies provide solid evidence for the potential of Niuchangchih to treat inflammation. To elucidate the mechanisms of action of antcin A and antrocamphin A in vivo, more animal experiments and randomized controlled clinical trials should be carried out. Since more than 20 compounds in Niuchangchih have been shown to possess anti-inflammatory activity6, 7, 8, further studies are needed to elucidate their mechanisms. It is also of interest to figure out whether these compounds act synergistically or independently. Notably, most anti-inflammatory compounds in Niuchangchih have been derived from the fruiting bodies so far. Further research is needed to uncover whether they can also be produced in fermented mycelia. Besides antcin A and antrocamphin A, many unknown ingredients responsible for the anti-infammatory effects of Niuchangchih remain to be discovered.

The present study investigated the three-dimensional spectra and emission spectra of the autofluorescence of rabbit hearts. The results suggested that the three-dimensional spectra of the iced atria and ventricle were observed more evidently different from that of the fresh tissue compared to the main artery, which indicated that the amount of flavins and NADHs changed. Also, the atria, ventricle and main artery have different specific excitation spectra at the wavelength of 340 nm. The main fluorescence peaks were of NADH (at about 460 nm), collagen and elastin (at about 290-400 nm). The Gauss spectra of atria and ventricle were different in the peak value, relative intensity and half width. So the ratios of fluorescence intensities of peaks may be used to distinguish different heart tissues. Furthermore, a phenomenon was firstly uncovered that the autofluorescence intensity of NADH in ventricle decays with the time of death and it could be a useful method for the estimation of postmortem interval.

The laser-inducted fluorescence spectrum technology (LIF) was used for the first time to study the autofluorescence spectral characteristics of the heart, kidney, liver, fat, foreleg muscle, hind leg soleus muscle and musculus gastrocnemius of the rat performing motion exercises. The wavelength of the excitation light used during the measurement was in the range of 250-650 nm and the emission wavelength was 300-700 nm. When comparing the three-dimensional fluorescence spectra of the control group with those of the four groups of different motion states, a specific fluorescence peak related to the motion and located in the area where the excitation wavelength was (340 +/- 10) nm and the emission wavelength was (460 +/- 10) nm was found mainly in the spectra of the soleus muscle. From this fluorescence peak, it is possible to determine that its corresponding fluorescent substance is NADH (nicotinamide adenine dinucleotide reduced). When comparing the fluorescence spectra of the four groups of different motion modes, it was found that the motion mode has a conspicuous relativity with the peak intensity. The results show that the energy metabolism of the soleus muscle of the rat in motion is stronger than that of the foreleg, soleus muscle and other visceras, and the autofluorescence spectral characteristics of NADH form one of the effective indexes for determining the muscular metabolism degree.

Recently, non-invasive optical methods to monitor transmembrane electrical potential using voltage sensitive dyes have been applied widely in the studies of normal and pathological heart rhythms and defibrillation. In the present paper, the authors measured the excitation and the emission spectra of the voltage-sensitive dyes di-4-ANEPPS bound to phospholipid bilayer membranes. And according to the spectral shift of di-4-ANEPPS, the authors presented an optical mapping system combining a DALSA CCD camera and a LED light source. Using this optical mapping system, the authors could record the action potential duration of the heart cells with high spatial and temporal resolutions. It can be a powerful tool in the study of cardiac arrhythmia mechanisms.

This study investigated the absorption spectrum and the fluorescence spectrum of rabbit hearts stained with the voltage sensitive dye (di-4-ANEPPS). The results suggested that the optical absorption of tissue with the dye was higher than that of the control group, and there were significant differences between the experimental group and control group in the range of 450-550 nm. It was also indicated that the maximum absorption peak of the dye in tissues was at (479.25 +/- 0.44) nm. Additionally, the different peaks of fluorescence emission spectra from the atriums, ventricles and aorta were originally found by testing the five parts of rabbit hearts with the dye. Their relative intensities were related to the distribution concentrations of the dye. Meanwhile, the peaks of excitation spectra and fluorescence emission spectra were determined by examining the three-dimensional and two-dimensional fluorescence spectra using ventricles and atriums with the dye. Based on the discrepancy of rest membrane voltages between ventricles and atriums, the best wavelength ranges of excitation light and emissions light of optical mapping were determined by the shifts of the dye in emission spectra with excitation at different wavelengths. These results offer a theoretical foundation for the design of a cardiac optical mapping system.

Microbial xylanases have received a great deal of attention in the last two decades for their potential applications in food, paper making and animal feed industries. Bacillus pumilus WL-11 was identified as a producer of alkane xylanase free of cellulase after screening soil samples of paper-making factories. The xylanase A (XylA) was purified to homogeneity from the culture filtrate of Bacillus pumilus WL-11 by (NH4) 2SO4 precipitation, CM-Sephadex and Sephadex G-75 chromatographies. The molecular mass of XylA is estimated to be 26.0 kD by SDS-PAGE and its isoelectric point is 9.5. The apparent Km is 16.6 mg/mL and V(max) is 1263 micromol/(min x mg) towards oat spelt xylan. XylA is optimally active between pH 7.2 and 8.0, and stable at pH 6.0 to 10.4. The enzyme is optimally active at 45 degrees C - 55 degrees C and stable at temperature below 45 degrees C, with its half time of activity of 35 min and 15 min at 55 degrees C and 60 degrees C respectively. HPLC analysis revealed that hydrolysis patterns of xylans from oat spelt, birch wood and beech wood by purified XylA were different. The XylA is determined to be an endo-beta-1,4-xylanase, as it generated mainly xylotriose and no xylose was detected among the three hydrolysates. XylA has strong hydrolytic activity towards the pentose in the hydrolysates of beech wood and birch wood xylans, but was not active to the pentose in the hydrolysate of oat spelt xylan. The crude WL-11 enzyme can efficiently hydrolyze oat spelt xylan to a series of xylo-oligosaccharides, suggesting its potential application in nutraceutical industry.

The psychrotrophs SYP-A2-3 producing the cold-adapted protease has been isolated from the bacterial samples collected from the No. 1 Glacier of China and identified as Bacillus cereus according to its morphological and physiochemical characteristics and 16s rDNA gene sequence analysis. It could grow between 0 degree C and 38 degrees C while its optimal growth temperature was 25 degrees C and the optimal temperature for its protease production was 15 degrees C. The cold-adapted protease was identified as neutral metallo-protease, the molecular weight was 34.2 kD shown by SDS-PAGE, the optimal pH and temperature for activity was 7.0-8.5 and 42 degrees C, respectively. Various fermentation conditions of its protease production were also investigated. The results showed that casein was the best nitrogen source while glucose and starch were suitable carbon source for its protease production. The initial pH of fermentation broth ranged from 6.5 to 7.0 was optimal. Under optimized conditions, the protease activity produced by SYP-A2-3 could reach 3800 U/mL and 4800 U/mL conducted in shaking flask and 5 L stirred jar experiment, respectively.

To study the stability of insulin-loaded polybutylcyanoacrylate nanoparticles (IPN) in an oily medium (soybean oil containing 0.5% (v/v) Tween-20 and 5% (v/v) Vitamin E) along with the hypoglycemic effect following their oral administration to streptozotocin (STZ) induced diabetic rats.The stability of IPN in the process was appraised by measurement of the amount of undegraded insulin associated to nanoparticles, the average size and the span of IPN, as well as the release of insulin from IPN. IPN in an aqueous medium (containing 0.5% (v/v) Tween-20) at pH 2.0 was also investigated as control.The study showed that IPN in the oily medium was more stable than that in the aqueous medium over one year of storage in the dark at (25 +/- 2) degrees C and the in vitro stability of IPN in the oily medium against degradation by proteolytic enzymes was much better than that in the aqueous medium. The apparent bioavailability of an oral administration of IPN (50 u x kg(-1)) in the oily medium versus an (sc) injection of insulin (2 u x kg(-1)) was 22.4%, much higher than that of IPN in the aqueous medium (15.5%), based on decreased areas above curve (AAC) determination for the blood glucose depression from time zero to 144 h of a single oral administration of IPN to STZ-diabetic rats.IPN in soybean oil containing Tween-20 (0.5% v/v) and Vitamin E (5% v/v) could be considered as an effective and stable delivery system for oral insulin.

An alkaline catalase has been purified and characterized from a slightly halophilic and alkaliphilic bacterium Bacillus sp. F26. The purification was performed with a four step procedure consisting of ammonium sulfate precipitation, ion exchange, gel filtration and hydrophobic interaction chromatography, and finally achieved a 58.5-fold-purifying over the crude extract. The purified catalase was composed of two identical subunits with a native molecular mass of 140 kD. The native enzyme showed the typical Soret band appearing at 408 nm. The pyridine hemochrome spectrum indicated the presence of protoheme IX as the prosthetic group. The apparent Km value for enzyme activity on H2O2 was calculated to be 32.5 mmol/L. The activity of this catalase was not reduced by dithionite but was strongly inhibited by cyanide, azide, and 3-amino-1,2,4-triazole (the specific inhibitor of monofunctional catalase). No peroxidase activity of this enzyme was detected when using o-dianisidine, diaminobenzidine (DAB) and p-phenylenediamine as electron donor. Moreover, the N-terminal sequence of this catalase exhibited substantial similarity to the monofunctional catalase subgroup rather than catalase-peroxidase or Mn-catalase one. Therefore, we characterize the purified catalase as a monofunctional catalase. Besides, this monofunctional catalase was thermosensitive and its activity exhibited pH-independent over pH 5-9 but showed a sharp maximum at pH 11. An activity half-life of approximately 49 h was measured when the enzyme was incubated at 20 degrees C and pH 11. To our knowledge, pH 11 is the most alkaline condition for optimum catalysis and enzyme stability among the catalases reported up to now. Furthermore, this monofunctional catalase also showed excellent halo-alkali-stability with a half-life of approximately 90 h at 0.5 mol/L NaCl and pH 10.5. On the other hand, so far as we know, the characterized catalase is the first dimeric monofunctional catalase from alkaliphiles and is also the first monofunctional catalase derived from a natural soda lake, which could partially reflect the oxidative stress response in the corresponding environment.