Sinomenine ameliorates adjuvant-induced arthritis by inhibiting the autophagy/NETosis/inflammation axis.

Studies have found that neutrophil extracellular traps (NETs) which are the specific dying form of neutrophil upon activation have fundamental role in the rheumatoid arthritis onset and progression. The purpose of this study was to explore the therapeutic effect of Sinomenine on adjuvant-induced arthritis in mice, and the neutrophil activities regulated by Sinomenine. The rheumatoid arthritis model was established by local injection of adjuvant and the Sinomenine treatment was administered orally for 30 days, during which, arthritic scores were evaluated and the joint diameter was measured to determine disease progression. The joint tissues and serum were acquired for further tests after sacrifice. Cytometric beads assay was performed to measure the concentration of cytokines. For paraffin-embedded ankle tissues, hematoxylin and erosin staining and Safranin O-fast staining were adopted to monitor the tissue changes of joint. In order to analyze the inflammation, NETs and autophagy of neutrophils in vivo, immunohistochemistry assays were applied to detect the protein expression levels in the local joints. To describe the effect brought by Sinomenine on inflammation, autophagy and NETs in vitro, the western blotting and the immunofluorescence assays were performed. The joint symptoms of the adjuvant induced arthritis were alleviated by the Sinomenine treatment significantly in terms of the ankle diameter and scores. The improvement of local histopathology changes and decrease of inflammatory cytokines in the serum also confirmed the efficacy. The expression levels of interleukin-6, P65 and p-P65 in the ankle areas of mice were remarkably reduced by Sinomenine. Compared with the model group, the decreased expression levels of lymphocyte antigen 6 complex and myeloperoxidase in the Sinomenine treating group showed the inhibitory effect of Sinomenine on the neutrophil migration. The expression of protein arginine deiminase type 4 (PAD4), ctrullinated histone H3 (CitH3) and microtubule-associated protein 1 light chain 3B (LC3B) had the similar tendency. Upon activation of lipopolysaccharide (LPS) in vitro, Sinomenine suppressed the phosphorylation of P65, extracellular signal-regulated kinase (ERK) and P38 of neutrophil. Meanwhile, Sinomenine inhibited NETs formation induced by phorbol 12-myristate 13-acetate (PMA), which were demonstrated by the decreased expression of neutrophil elastase (NE), PAD4 and CitH3. Sinomenine also inhibited PMA-induced autophagy in vitro based on the changes of Beclin-1 and LC3B. Sinomenine has good efficacy in treating adjuvant induced arthritis via regulating neutrophil activities. Apart from inhibiting activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, the mechanism includes suppression of NETs formation via autophagy inhibition.

Synthesis and structure-activity relationship of oleanolic mono- or di-glycosides against Magnaporthe oryzae.

Saponins are naturally-occurring units with broad diversity and are usually recognized as phytoanticipins. In order to develop new saponin chemical entities with high activity against Magnaporthe oryzae, we selected oleanolic acid (OA), which has wide natural distribution and rich content in plants. We used the ability of OA to act as an aglycone for glycosylation to obtain information on the structure-activity relationship (SAR) for rational molecular pesticide design. Oleanolic mono- or di-glycosides were synthesized at either the C3-hydroxy and/or C28-carboxyl position, using trichloroacetimidate or glycosyl bromide donors, respectively. Structures were confirmed by [1H]-,[13C]-NMR. Furthermore, the activity of the synthesized glycosides against M. oryzae was assessed in vitro, based on the mycelium growth rate. The twenty five oleanolic mono- or di-glycosides comprised fourteen saponins with 3-monosaccharide residue 1a-1n, six saponins with 28-monosaccharide residue 2a-2f, and five saponins with 3, 28-monosaccharide residue 3a-3e; all showed different activities against M. oryzae according to their different structures. We concluded that the optimal oleanolic mono- and di-glycoside structure for activity against M. oryzae is a C3 connection of a hexose such as mannose, galactose, or glucose, in combination with a C28 connection to a small group such as allyl or a C3 connection to a pentose accompanied by a larger group such as another pentose or heptenyl at C28.

ß-Galactosidase with transgalactosylation activity from Lactobacillus fermentum K4.

The LacLM ß-galactosidase of Lactobacillus fermentum K4 is encoded by 2 consecutive genes, lacL (large subunit) and lacM (small subunit), that share 17 overlapping nucleotides. Phylogenetic analysis revealed that this enzyme was closely related to other Lactobacillus ß-galactosidases and provided significant insight into its common and distinct characteristics. We cloned both the lacL and lacM genes of L. fermentum K4 and heterologously expressed each in Escherichia coli, although the recombinant enzyme was only functional when both were expressed on the same plasmid. We evaluated the enzymatic properties of this species-specific LacLM ß-galactosidase and discovered that it acts as both a hydrolase, bioconverting lactose into glucose and galactose, and a transgalactosylase, generating prebiotic galacto-oligosaccharides (GOS). The recombinant ß-galactosidase showed a broad pH optimum and stability around neutral pH. The optimal temperature and Michaelis constant (K(m)) for the substrates o-nitrophenyl-ß-D-galactopyranoside and lactose were, respectively, 40°C and 45 to 50°C and 1.31 mM and 27 mM. The enzyme activity was stimulated by some cations such as Na⁺, K⁺, and Mg²âº. In addition, activity was also enhanced by ethanol (15%, wt/vol). The transgalactosylation activity of L. fermentum K4 ß-galactosidase effectively and rapidly generated GOS, up to 37% of the total sugars from the reaction. Collectively, our results suggested that the ß-galactosidase from L. fermentum K4 could be exploited for the formation of GOS.

Nanosize structural modifications with polarization functions in ultrafast laser irradiated bulk fused silica.

Laser-induced self-organization of regular nanoscale layered patterns in fused silica is investigated using spectroscopy and microscopy methods, revealing a high presence of stable broken oxygen bonds. Longitudinal traces are then generated by replicating static irradiation structures where the nanoscale modulation can cover partially or completely the photoinscribed traces. The resulting birefringence, the observed anisotropic light scattering properties, and the capacity to write and erase modulated patterns can be used in designing bulk polarization sensitive devices. Various laser-induced structures with optical properties combining guiding, scattering, and polarization sensitivity are reported. The attached polarization functions were evaluated as a function of the fill factor of the nanostructured domains. The polarization sensitivity allows particular light propagation and confinement properties in three dimensional structures.

Adhesion capability of first two domains at N terminus of NP_785232 protein and their interaction with a UV-absorbing component from human mucus.

AIMS: This work aims to investigate the binding capability of certain domains at N terminus of the protein NP_785232 of Lactobacillus plantarum to Caco-2 cells and to test the usage of affinity chromatography to isolate the human mucus component that interacts with them. METHODS AND RESULTS: Recombinant proteins containing the first and both the first and second domains at N terminus of NP_785232 fused to a His tag were constructed and used to bind the Caco-2 cells. The interacting molecule from human mucus was isolated by affinity chromatography through immobilizing the recombinant proteins onto a Sepharose matrix. It was found both recombinant proteins could block the adhesion of Lact. plantarum to Caco-2 cells and bind to a human mucus component. CONCLUSIONS: The first and both the first and second domains at N terminus of the protein NP_785232 have the capability to adhere Caco-2 cells and by affinity chromatography, an interacting UV-absorbing component from human mucus was isolated. SIGNIFICANCE AND IMPACT OF THE STUDY: The protein domains characterized in this study may be displayed on probiotics to promote adhesion, and further characterization of the human mucus component might be helpful to identify host factors required for prolonging probiotics persistence in the gastrointestinal tract.

Monoclonal antibodies against Stx1B subunit of Escherichia coli O157:H7 distinguish the bacterium from other bacteria.

AIMS: The Shiga-like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA. METHODS AND RESULTS: Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non-Stx1-producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus. CONCLUSIONS: The anti-STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report regarding the usage of anti-STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection.

Expression of milk-derived antihypertensive peptide in Escherichia coli.

Many angiotensin converting enzyme inhibitory peptides (ACEIP) have been identified in recent years. Among all the literatures available thus far, almost all the ACEIP were obtained by means of enzymatic hydrolysis. However, little information was available on antihypertensive peptides obtained by DNA recombination technology. In the present paper, our aims were 1) to establish a new method to produce antihypertensive peptides (AHP), and 2) to study the expression profiles of different host strains (Escherichia coli JM109 and DH5alpha). To achieve these objectives, a DNA fragment encoding the published ACEIP, identified as FFVAPFPEVFGK (known as CEI12) was synthesized, ligated with the expression vector, pQE16, and transformed into E. coli JM109 and DH5alpha. SDS-PAGE analysis and Western-blotting detection demonstrated that the peptide CEI12 (fused with dihydrofolate reductase [DHFR]) was specifically expressed only in E. coli JM109 with IPTG induction. The expression profiles of the AHP CEI12 at different IPTG concentrations and different inducing times demonstrated no significant differences by SDS-PAGE analysis. The expression level of CEI12 (fused with DHFR) was about 500 microg/L culture.

[Clinical study on treatment of chronic bronchitis by tracheitis plaster].

OBJECTIVE: To study the clinical effect and mechanism of Tracheitis Plaster (TP) in treating chronic bronchitis. METHODS: TP is consisted of ephedra, almond, pinellia tuber, earthworm and white mustard seed. Patients were randomly divided into two groups, 59 patients in the treated group were treated with TP sticking on acupoints Dingchuan, Dashu, Fengmen, Feishu and Xinshu at back along both sides of thoracic vertebrae 1-6 and the 25 patients in the control group were treated with intramuscular injection of Siqikang. The times of treatment for both groups were 20. Clinical symptoms, X-ray chest film, level of immunoglobulin and T-lymphocyte subsets were recorded before and after treatment, and follow-up were carried out 0.5-1 year later. RESULTS: The clinical total effective rate was 93.2% and the X-ray improvement rate was 40.7% in the treated group, while in the control group, 80.0% and 20.0% respectively. Half and 1 year follow-up studies showed the total effective rate in the treated group was 91.5% and 89.8% respectively, which was significantly higher than that in the control group (80.0% and 76.0%) respectively (P < 0.05). The improvement in levels of IgG and CD8 in the treated group was also superior to those in the control group (P < 0.05). CONCLUSION: TP is a highly effective transcutaneous absorbent with promising long-term effect, it could regulate the immune function.

[Determination of trace copper and lead in highly pure samaria by extraction-graphite furnace atomic absorption spectrometry].

In this paper, we have studied the meassuring condition and the extracting condition for the determination of trace copper and lead in highly pure samaria by extraction-GFAAS. Trace copper and lead in highly pure samaria were determined. The relative standard deviations are less than 6%, the recovery is 98-101% for copper and 93-100% for lead. The method is simple, accurate and practical.

[Experimental study on the influence of sodium glutamate on embryonal brain development of mouse].

The main component of gourment powder is a typical excitatory amino acid. Its neurotoxic action has been attended extensively. Healthy Kunming strain mice were used in this study. Sodium glutamate was injected into peritoneal cavity from the fourth day of gestation (at dosages of 60, 30 or 10 mg/kg every other day). The embryonal mice were dissected at the eighteenth day of gestation. The brain of embryonal mouse was removed, weighted, and fixed in Carnory's solution. Routine paraffin section was made. HE, Feulgen and Nissl staining methods were used for morphological observation. The ash values of 100 neurons of cerebral cortex of Feulgen and Nissl staining were randomly measured with an automated image analyser. The results showed that there was no morphological change in the experimental and control group. There was no significant differece between experimental groups and control group in the parameters observed (P > 0.05). It was suggested that sodium glutamate was not harmful to embryonal brain development of mouse.