Synthesis and evaluation of cytotoxic effects of hanultarin and its derivatives

One of the known cytotoxic lignans is (-)-1-O-feruloyl-secoisolariciresinol designated as hanultarin, which was isolated from the seeds of Trichosanthes kirilowii. In this Letter, we described the first synthesis of 1-O-feruloyl-secoisolariciresinol, 1,4-O-diferuloyl-secoisolariceresinol and their analogues. The cytotoxicities of these compounds were evaluated against several cancer cell lines. Interestingly, we found that the feruloyl diester derivative of secoisolariciresinol was the most active cytotoxic compound against all the cancer cells tested in this experiment. The IC(50) values of the1,4-O-diferuloyl-secoisolariceresinol were in the range of 7.1-9.8μM except one cell line. In considering that both ferulic acid and secoisolariciresinol are commonly found in many plants and have no cytotoxicity, this finding is remarkable in that simple covalent bonds between the ferulic acid and secoisolariciresinol cause a cytotoxic effect.     

Low‐generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA

Conventional dendrimers are spherical symmetrically branched polymers ending with active surface functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, higher‐generation (G4‐G8) PAMAM dendrimers exhibit toxicity due to their high cationic charge density and this has limited their application in vitro and in vivo. Another limitation arises when attempts are made to functionalize spherical dendrimers as targeting moieties cannot be site‐specifically attached. Therefore, we propose that lower‐generation asymmetric dendrimers, which are likely devoid of toxicity and to which site‐specific attachment of targeting ligands can be achieved, would be a viable alternative to currently available dendrimers. We synthesized and characterized a series of peptide‐based asymmetric dendrimers and compared their toxicity profile and ability to condense DNA to spherical PAMAM G1 dendrimers. We show that asymmetric dendrimers are minimally toxic and condense DNA into stable toroids which have been reported necessary for efficient cell transfection. This paves the way for these systems to be conjugated with targeting ligands for gene delivery in vitro and in vivo. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Ecosystem Restoration of Jhum Fallows in Northeast India: Microbial C and N Along Altitudinal and Successional Gradients

Abstract To characterize the altitudinal and successional trends in microbial biomass and to understand their role in soil nutrient dynamics during the aggradation phase (vegetation recovery) of abandoned shifting cultivation systems, we determined the soil properties and microbial C and N in jhum (slash‐and‐burn) cultivation systems at different altitudes and 1‐, 7‐, and 16‐year‐old fallow agricultural lands at lower and higher altitudes in the northeastern Indian hills. Density of ground vegetation was lower in the undisturbed forest than in the jhum fallows. In general, 1‐year jhum fallow had greater herbaceous vegetation both at lower and higher altitudes. Although woody plants were observed in 7‐ and 16‐year‐old jhum fallows, their density was highest in the forest. Soil moisture, organic C, and total N also increased gradually with increasing altitude and progressive secondary succession. Soil pH showed a negative correlation with altitude (as also confounded by soil type) and fallow age. Both microbial C and N had a close correlation with altitude and fallow age. Contribution of microbial C to soil organic C was 2.0–2.6% and microbial N to total N 1.4–2.2% in jhum fields, 2.4–4.3% and 1.2–2.1%, respectively, in jhum fallows, and 2.5–2.9% and 1.6–1.9% in the forests. Microbial C and N showed a negative correlation with herbaceous plant density. Microbial biomass in the jhum fallows and forest stands had a positive relationship with woody vegetation. Along an altitudinal and/or successional gradient, microbial C and N were positively correlated with water‐holding capacity, soil moisture, organic C, and total N and negatively correlated with soil pH. Microbial C and N were positively correlated with each other. Therefore, the study suggests that the altitudinal and successional dynamics of microbial C and N are linked to, among other properties, soil organic matter and total nitrogen contents in the soil during community development after land abandonment from shifting cultivation.     

The effect of pH on PAMAM dendrimer-siRNA complexation: endosomal considerations as determined by molecular dynamics simulation

Intracellular degradation of genes, most notably within the endo-lysosomal compartment is considered a significant barrier to (non-viral) gene delivery in vivo. Previous reports based on in vitro studies claim that carriers possessing a mixture of primary, secondary and tertiary amines are able to buffer the acidic environment within the endosome, allowing for timely release of their contents, leading to higher transfection rates. In this report, we adopt an atomistic molecular dynamics (MD) simulation approach, comparing the complexation of 21-bp siRNA with low-generation polyamidoamine (PAMAM) dendrimers (G0 and G1) at both neutral and acidic pHs, the latter of which mimics the degradative environment within maturing 'late-endosomes'. Our simulations reveal that the time taken for the dendrimer-gene complex (dendriplex) to reach equilibrium is appreciably longer at low pH and this is accompanied by more compact packaging of the dendriplex, as compared to simulations performed at neutral pH. We also note larger absolute values of calculated binding free energies of the dendriplex at low pH, indicating a higher dendrimer-nucleic acid affinity in comparison with neutral pH. These novel simulations provide a more detailed understanding of low molecular-weight polymer-siRNA behavior, mimicking the endosomal environment and provide input of direct relevance to the "proton sponge theory", thereby advancing the rational design of non-viral gene delivery systems.     

Characterization of Programmed Death-1 Homologue-1 (PD-1H) Expression and Function in Normal and HIV Infected Individuals

Chronic immune activation that persists despite anti-retroviral therapy (ART) is the strongest predictor of disease progression in HIV infection. Monocyte/macrophages in HIV-infected individuals are known to spontaneously secrete cytokines, although neither the mechanism nor the molecules involved are known. Here we show that overexpression of the newly described co-stimulatory molecule, PD1 homologue (PD-1H) in human monocyte/macrophages is sufficient to induce spontaneous secretion of multiple cytokines. The process requires signaling via PD-1H as cytokine secretion could be abrogated by deletion of the cytoplasmic domain. Such overexpression of PD-1H, associated with spontaneous cytokine expression is seen in monocytes from chronically HIV-infected individuals and this correlates with immune activation and CD4 depletion, but not viral load. Moreover, antigen presentation by PD-1H-overexpressing monocytes results in enhanced cytokine secretion by HIV-specific T cells. These results suggest that PD-1H might play a crucial role in modulating immune activation and immune response in HIV infection.     

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.