Optimization of culture media for enhanced chitinase production from a novel strain of Stenotrophomonas maltophilia using response surface methodology

Chitinase is one of the most important mycolytic enzymes with industrial significance. This enzyme is produced by a number of organisms including bacteria. In this study we describe optimization of media components with increased production of chitinase for selected bacteria Stenotrophomonas maltophilia isolated from the soil. Different components of the defined media responsible for influencing chitinase secretion by the bacterial isolate were screened using Plackett-Burman experimental design and were further optimized by Box-Behnken factorial design of response surface methodology (RSM) in liquid culture. Maximum chitinase production was predicted in medium containing chitin 4.94 g/l, maltose 5.56 g/l, yeast extract 0.62 g/l, KH2PO4 1.33 g/l and MgSO4.7H2O 0.65 g/l using Response surface plots and point prediction tool of DESIGN EXPERT 7.1.6 (Statease, USA) software.     

Identification of a sulfonoquinovosyldiacylglyceride from Azadirachta indica and studies on its cytotoxic activity and DNA binding properties

Chromatographic separation of the methanolic extract of the leaves of Azadirachta indica led to the isolation of a sulfonoglycolipid characterized as a sulfonoquinovosyldiacylglyceride (SQDG), by extensive 2D NMR and mass spectral analysis. SQDG induces apoptosis in a dose dependent manner with IC(50) 8.3 μM against acute lymphoblastic leukemia (ALL) MOLT-4 cell lines. The compound showed significant DNA binding properties as evidenced by the enhancement of melting temperature and perturbation of the characteristic B-form in CD evidence of calf thymus DNA. The DNA binding was also characterized by isothermal calorimetry where a predominantly enthalpy driven binding to CT DNA was revealed.     

Phagocytosis Is the Main CR3-Mediated Function Affected by the Lupus-Associated Variant of CD11b in Human Myeloid Cells

The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis.     

Mild and efficient synthesis of new tetraketones as lipoxygenase inhibitors and antioxidants

A mild and efficient route to tetraketones (2–22) has been developed by way of tetraethyl ammonium bromide (Et₄N⁺Br^? ) mediated condensation of dimedone (5,5-dimethylcyclohexane-1,3-dione, 1) with a variety of aldehydes. All these compounds showed significant lipoxygenase inhibitory activity and moderate to strong antioxidant potential. Compounds 19 (IC₅₀ = 7.8 μM), 22 (IC₅₀ = 12.5 μM), 3 (IC₅₀ = 16.3 μM), 11 (IC₅₀ = 17.5 μM) and 8 (IC₅₀ = 21.3 μM) showed significant inhibitory potential against lipoxygenase (baicalein, IC₅₀ = 22.4 μM). On the other hand compound 19 (IC₅₀ = 33.6 μM) also showed strong antioxidant activity compared to the standard (IC₅₀ = 44.7 μM). This study is likely to lead to the discovery of therapeutically efficient agents against very important disorders including inflammation, asthma, cancer and autoimmune diseases.     

Urease and Serine Protease inhibitory alkaloids from Isatis tinctoria

Phytochemical investigations on the alkaloidal fraction of the whole plant of the Isatis tinctoria led to the isolation of the alkaloids 1-6., 3′-Hydroxyepiglucoisatisin (3), Epiglucoisatisin (2) were found to be potent urease inhibitors in a concentration-dependent manner with IC₅₀ values 25.63 ± 0.74, 37.01 ± 0.41 and 31.72 ± 0.93, 47.33 ± 0.31 μM against Bacillus pasteurii & Jack bean urease, respectively. Compounds 3 and 2 also showed potent inhibitory potential against α-chymotrypsin with IC₅₀ values of 23.40 ± 0.21 and 27.45 ± 0.23 μM, respectively.     

Evaluation of In Vitro Cross-Reactivity to Avian H5N1 and Pandemic H1N1 2009 Influenza Following Prime Boost Regimens of Seasonal Influenza Vaccination in Healthy Human Subjects: A Randomised Trial

Introduction

Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses.

Methods

In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose.

Results

Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated.

Conclusion

Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01044095","term_id":"NCT01044095"}}NCT01044095