Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. Frutapin (FTP) is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically-active recombinant FTP in E. coli BL21, optimizing conditions with the best set yielding >40 mg/L culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/mL of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP-mannose and FTP-glucose crystals were obtained and diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å, respectively. The best solution showed four monomers per asymmetric unit. Molecular Dynamics simulation suggested FTP displays higher affinity for mannose than glucose. Cell studies revealed FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/mL and also capable of stimulating cell migration at 50 µg/mL. In conclusion, our optimized expression system allowed high amounts of correctly-folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example, in wound healing and tissue regeneration

Processes Controlling Tropical Tropopause Temperature and Stratospheric Water Vapor in Climate Models

A warm bias in tropical tropopause temperature is found in the Met Office Unified Model (MetUM), in common with most models from phase 5 of CMIP (CMIP5). Key dynamical, microphysical, and radiative processes influencing the tropical tropopause temperature and lower-stratospheric water vapor concentrations in climate models are investigated using the MetUM. A series of sensitivity experiments are run to separate the effects of vertical advection, ice optical and microphysical properties, convection, cirrus clouds, and atmospheric composition on simulated tropopause temperature and lower-stratospheric water vapor concentrations in the tropics. The numerical accuracy of the vertical advection, determined in the MetUM by the choice of interpolation and conservation schemes used, is found to be particularly important. Microphysical and radiative processes are found to influence stratospheric water vapor both through modifying the tropical tropopause temperature and through modifying upper-tropospheric water vapor concentrations, allowing more water vapor to be advected into the stratosphere. The representation of any of the processes discussed can act to significantly reduce biases in tropical tropopause temperature and stratospheric water vapor in a physical way, thereby improving climate simulations

Evaluation of the genotoxic and antigenotoxic potential of Melissa officinalis in mice

Melissa officinalis (L.) (Lamiaceae), a plant known as the lemon balm, is native to the east Mediterranean region and west Asia. Also found in tropical countries, such as Brazil, where it is popularly known as “erva-cidreira” or “melissa”, it is widely used in aqueous- or alcoholic-extract form in the treatment of various disorders. The aim was to investigate in vivo its antigenotoxicity and antimutagenicity, as well as its genotoxic/mutagenic potential through comet and micronucleus assaying. CF-1 male mice were treated with ethanolic (Mo-EE) (250 or 500 mg/kg) or aqueous (Mo-AE) (100 mg/kg) solutions of an M. officinalis extract for 2 weeks, prior to treatment with saline or Methyl methanesulfonate (MMS) doses by intraperitoneal injection. Irrespective of the doses, no genotoxic or mutagenic effects were observed in blood and bone-marrow samples. Although Mo-EE exerted an antigenotoxic effect on the blood cells of mice treated with the alkylating agent (MMS) in all the doses, this was not so with Mo-AE. Micronucleus testing revealed the protector effect of Mo-EE, but only when administered at the highest dose. The implication that an ethanolic extract of M. officinalis has antigenotoxic/antimutagenic properties is an indication of its medicinal relevance

An “Escape Clock” for Estimating the Turnover of SIV DNA in Resting CD4+ T Cells

Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an “escape clock” approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT) SIV DNA present in early infection by CTL escape mutant (EM) strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication