Novel Aporphine- and Proaporphine-Clerodane Hybrids Identified from the Barks of Taiwanese Polyalthia longifolia (Sonn.) Thwaites var. pendula with Strong Anti-DENV2 Activity.

Hybrid natural products produced via mixed biosynthetic pathways are unique and often surprise one with unexpected medicinal properties in addition to their fascinating structural complexity/diversity. In view of chemical structures, hybridization is a way of diversifying natural products usually through dimerization of two similar or dissimilar subcomponents through a C-C or N-C covalent linkage. Here, we report four structurally attractive diterpene-alkaloid conjugates polyalongarins A-D (1-4), clerodane-containing aporphine and proaporphine alkaloids, the first of its kind from the barks of Taiwanese Polyalthia longifolia (Sonn.) Thwaites var. pendula. In addition to conventional spectroscopic analysis, single crystal X-ray crystallography was employed to determine the chemical structures and stereo-configurations of 1. Compounds 1-4 were subsequently subjected to in vitro antiviral examination against DENV2 by evaluating the expression level of the NS2B protein in DENV2-infected Huh-7 cells. These compounds display encouraging anti-DENV2 activity with superb EC50 (2.8-6.4 µM) and CC50 values (50.4-200 µM). The inhibitory mechanism of 1-4 on NS2B was further explored drawing on in-silico molecular docking analysis. Based on calculated binding affinities and predicted interactions between the functional groups of 1-4 and the allosteric-site residues of the DENV2 NS2B-NS3 protease, our analysis concludes that the clerodane-aporphine/proaporphine-type hybrids are novel and effective DENV NS2B-NS3 protease inhibitors.

Protective Immunity against Vibrio harveyi in Grouper Induced by Single Vaccination with Poly (Lactide-co-glycolide) Microparticles Releasing Pleurocidin Peptide and Recombinant Glyceraldehyde-3-phosphate Dehydrogenase.

The peptide adjuvant, pleurocidin (PLE), and the Vibrio harveyi antigen, recombinant glyceraldehyde-3-phosphate dehydrogenase (rGAPDH) protein, were encapsulated with poly (lactide-co-glycolide) (PLG) polymers in our previous study to produce PLG-encapsulated PLE plus rGAPDH microparticles (PLG-PLE/rGAPDH MPs) that sustained stable release of both PLE and rGAPDH as well as, after two-time vaccination with MPs, generated long-term protective immunity against V. harveyi in grouper. Stable controlled-release of PLE plus rGAPDH from PLG-PLE/rGAPDH MPs is an attractive feature for developing an effective single-dose vaccine. In the present study, therefore, we aim to evaluate whether single administration with PLG-PLE/rGAPDH MPs in grouper would result in protective immunity against V. harveyi. Peritoneal vaccination of grouper with one dose of PLG-PLE/rGAPDH MPs raised serum titers over a long 12-week period. Moreover, twelve weeks after vaccination, significant lymphocyte proliferation and maximum TNF-α production were found in grouper immunized with a single dose of PLG-PLE/rGAPDH MPs. More importantly, immune responses elicited by single vaccination with PLG-PLE/rGAPDH MPs protected 80% of fish against a lethal peritoneal challenge of the highly virulent V. harveyi (Vh MML-1). In conclusion, our data truly reveal the feasibility of the development of a single-dose vaccine against V. harveyi based on PLG-PLE/rGAPDH MPs.

Warm SPA-induced hyperthermia confers protection to rats against airway inflammation evoked by capsaicin and substance P.

Solus par aqua (SPA) is a traditional health care therapy. Warm SPA may enhance immunity and cellular defense to protect body against diseases. The present study investigated whether the warm SPA could confer protection to neurogenic inflammation in rats. The rats were immersed in water where the body core temperatures were maintained at hyperthermia (41.5 degrees C) or normothermia (37 degrees C) for a period of 15min. After SPA for 1 or 6 days, neurogenic inflammation was induced by intravenous injection of capsaicin (90microg/kg) or substance P (SP; 3microg/kg). The plasma leakage and arterial pressures in rats after neurogenic inflammation were monitored. The extent of capsaicin- or SP-induced plasma leakage and hypotension was significantly attenuated in rats on day 1 after SPA hyperthermia. However, such resistance to neurogenic inflammation was not found on day 6 after hyperthermia. Western blotting analysis showed that the expression of heat shock protein 72 (HSP 72) in the trachea on days 1 and 2 after hyperthermia was 9.61-fold and 6.66-fold, respectively, of that in normothermia. Afterwards, the hyperthermia-induced HSP 72 upregulation gradually declined in a time-dependent manner. Thus, SPA hyperthermia may protect rats against neurogenic inflammation through modulation of HSP expression.

Dimethylthiourea pretreatment inhibits endotoxin-induced compound exocytosis in goblet cells and plasma leakage of rat small intestine.

Intravenous application of a high dose of endotoxin, also called lipopoly-saccharide (LPS), results in endotoxemia in animals, that induces production of cytokines and free radicals, systemic inflammation and mucin discharge from mucous tissues. The present study was to investigate (1) whether LPS application increased goblet cell secretion by compound exocytotic activity in mucosal villi and crypts of rat small intestine, and (2) whether hydroxyl radicals were involved in LPS-induced compound exocytosis in goblet cells and plasma leakage. Scanning electron microscopy showed that the numbers of goblet cells undergoing compound exocytosis (cavitated goblet cells) per mm(2) of ileal villus epithelium in rats 5 and 30 min after LPS (15 mg kg(-1)) were 693 +/- 196 (N = 6) and 547 +/- 213 (N = 6), respectively, which were 5.1 and 8.4 times (P < 0.05) the number of saline control. The percentage of villus cavitated goblet cell numbers, in both duodenum and ileum 5 min after LPS and in the ileum 30 min after LPS, increased significantly (P < 0.05). Pretreatment with dimethylthiourea (DMTU), a hydroxyl radical scavenger, decreased the number of cavitated goblet cells to saline control (P > 0.05). Morphometric analysis showed that the percentage of crypt epithelial area in the duodenum and ileum occupied by goblet cell mucin stores in the duodenum and ileum 30 min after LPS were 3.8 +/- 0.2% (N = 6) and 6.9 +/- 0.5 (N = 6), respectively reducing to one half the amount of control (P < 0.01). When DMTU was given prior to LPS the crypt goblet cell mucin stores and the amount of plasma leakage returned to the level of control. It is concluded that hydroxyl radicals were involved in the LPS-induced increase in compound exocytotic activity of goblet cells and the increase in plasma leakage during acute phases of inflammatory response in rat small intestine.

Inhibitory effect of dimethylthiourea on rat urinary bladder inflammation produced by 6-hydroxydopamine application.

The present study was to investigate 6-hydroxydopamine (6-OHDA)-induced inflammatory response and underlying mechanisms in the urinary bladder in anesthetized male rats of Long-Evans strain. The magnitude of inflammation was evaluated by morphometric analysis of the relative number of leaky blood vessels expressed by the area density of India ink-labeled blood vessels in whole mount specimens. Light and scanning electron microscopies were employed to study the changes in histologic structure and endothelial ultrastructure of bladder wall. Local injection of 6-OHDA to lumen of urinary bladder induced a dose-dependent increase in plasma leakage. Following application of vehicle, 5 mg/kg 6-OHDA, and 10 mg/kg 6-OHDA, area densities of India ink-labeled leaky vessels were 5.65+/-3.72% (N=6), 22.63+/-5.12% (N=6), and 35.02+/-11.25% (N=6), respectively. Inflammatory response was completely abolished by pretreatment alone with dimethylthiourea (DMTU), a hydroxyl radical scavenger, and was also attenuated by pretreatment with L-732,138, a NK1 receptor antagonist. 6-OHDA caused edema formation and venular endothelial gap formation in bladder tissue. It is concluded that 6-OHDA induced inflammation in the rat urinary bladder, the response of which was dose-dependently increased and free radicals and tachykinins were involved in the inflammatory process.