Antagonistic control of a dual-input mammalian gene switch by food additives

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.     

Synthesis of novel acridine and bis acridine sulfonamides with effective inhibitory activity against the cytosolic carbonic anhydrase isoforms II and VII

4-Amino-N-(4-sulfamoylphenyl)benzamide was synthesized by reduction of 4-nitro-N-(4-sulfamoylphenyl)benzamide and used to synthesize novel acridine sulfonamide compounds, by a coupling reaction with cyclic-1,3-diketones and aromatic aldehydes. The new compounds were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), and more precisely the cytosolic isoforms hCA I, II and VII. hCA I was inhibited in the micromolar range by the new compounds (K_Is of 0.16–9.64 μM) whereas hCA II and VII showed higher affinity for these compounds, with K_Is in the range of 15–96 nM for hCA II, and of 4–498 nM for hCA VII. The structure–activity relationships for the inhibition of these isoforms with the acridine–sulfonamides reported here were also elucidated.     

Selenium addition alters mercury uptake,bioavailability in the rhizosphere and root anatomy of rice (Oryza sativa)

Background and Aims

Mercury (Hg) is an extremely toxic pollutant, especially in the form of methylmercury (MeHg), whereas selenium (Se) is an essential trace element in the human diet. This study aimed to ascertain whether addition of Se can produce rice with enriched Se and lowered Hg content when growing in Hg-contaminated paddy fields and, if so, to determine the possible mechanisms behind these effects.

Methods

Two cultivars of rice (Oryza sativa, japonica and indica) were grown in either hydroponic solutions or soil rhizobags with different Se and Hg treatments. Concentrations of total Hg, MeHg and Se were determined in the roots, shoots and brown rice, together with Hg uptake kinetics and Hg bioavailability in the soil. Root anatonmy was also studied.

Key Results

The high Se treatment (5 μg g^–1) significantly increased brown rice yield by 48 % and total Se content by 2·8-fold, and decreased total Hg and MeHg by 47 and 55 %, respectively, compared with the control treatments. The high Se treatment also markedly reduced ‘water-soluble’ Hg and MeHg concentrations in the rhizosphere soil, decreased the uptake capacity of Hg by roots and enhanced the development of apoplastic barriers in the root endodermis.

Conclusions

Addition of Se to Hg-contaminated soil can help produce brown rice that is simultaneously enriched in Se and contains less total Hg and MeHg. The lowered accumulation of total Hg and MeHg appears to be the result of reduced bioavailability of Hg and production of MeHg in the rhizosphere, suppression of uptake of Hg into the root cells and an enhancement of the development of apoplastic barriers in the endodermis of the roots.     

Cellulose microfibril angle variation in Picea crassifolia tree rings improves climate signals on the Tibetan plateau

Microfibril angle (MFA) is an important factor in determining the mechanical properties of individual cells and wood as a whole. While some studies have described the variation of MFA within trees, little work has been done on the extent to which MFA is influenced by climate, despite it being known to respond to climatic events. Year-to-year variation in MFA and ring width was measured at high resolution by SilviScan-3^? on 30 dated Picea crassifolia trees growing in the northeastern Tibetan plateau. The climate signals registered in MFA and ring width were analyzed using dendroclimatological methods. The response function of MFA accounted for 67% of total variance, of which 60% was explained by climate elements. The response function of ring width explained 57% total variance, 37% of which was explained by climate variables. MFA significantly responded to July–August temperatures, and to precipitation in March, May and September. Over the period 1987–2009 temperatures generally increase and appeared to have a greater influence on MFA. A decrease in the strength of the relationship between MFA and ring width over the period 1987–2009 was also observed. MFA offers the potential to build robust climate proxies. The strong climate sensitivity of MFA to increasing temperature or the observed changes in the MFA–ring width relationship may contribute to resolving the “divergence problem” in temperature reconstructions. As far as we are aware, this study is the first to show a strong climate response in MFA and suggests that it might be a useful climate proxy.     

Direct detection of beta-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoelectrofocusing gels

A procedure to assay isozymes of beta-1,3-glucanase directly on polyacrylamide gel electrophoresis (PAGE) and isoelectrofocusing (IEF) gels by using 2,3,5-triphenyltetrazolium chloride is described. The reagent reacts with reducing sugars released by beta-1,3-glucanases from the substrate laminarin. Acidic and neutral isozymes of beta-1,3-glucanase were detected and quantified on 17.5% native PAGE gels run with an anodic buffer system. A significant linear relationship (alpha = less than 0.01, R = 0.991) was observed between amounts of beta-1,3-glucanase loaded and intensity of bands stained with the reagent on native PAGE gels. A full isozyme pattern was obtained on 7.5% IEF gels with a pH range of 3.5-9.5. The IEF gels were heated in a microwave oven during the staining process to minimize diffusion.     

3-(7-Azaindolyl)-4-arylmaleimides as potent, selective inhibitors of glycogen synthase kinase-3

A novel series of acyclic 3-(7-azaindolyl)-4-(aryl/heteroaryl)maleimides was synthesized and evaluated for activity against GSK-3beta and selectivity versus PKC-betaII, as well as a broad panel of protein kinases. Compounds 14 and 17c potently inhibited GSK-3beta (IC(50)=7 and 26 nM, respectively) and exhibited excellent selectivity over PKC-betaII (325 and >385-fold, respectively). Compound 17c was also highly selective against 68 other protein kinases. In a cell-based functional assay, both 14 and 17c effectively increased glycogen synthase activity by inhibiting GSK-3beta.     

Mutations at the S1 sites of methionine aminopeptidases from Escherichia coli and Homo sapiens reveal the residues critical for substrate specificity

Methionine aminopeptidase (MetAP) catalyzes the removal of methionine from newly synthesized polypeptides. MetAP carries out this cleavage with high precision, and Met is the only natural amino acid residue at the N terminus that is accepted, although type I and type II MetAPs use two different sets of residues to form the hydrophobic S1 site. Characteristics of the S1 binding pocket in type I MetAP were investigated by systematic mutation of each of the seven S1 residues in Escherichia coli MetAP type I (EcMetAP1) and human MetAP type I (HsMetAP1). We found that Tyr-65 and Trp-221 in EcMetAP1, as well as the corresponding residues Phe-197 and Trp-352 in HsMetAP1, were essential for the hydrolysis of a thiopeptolide substrate, Met-S-Gly-Phe. Mutation of Phe-191 to Ala in HsMetAP1 caused inactivity in contrast to the full activity of EcMetAP1(Y62A), which may suggest a subtle difference between the two type I enzymes. The more striking finding is that mutation of Cys-70 in EcMetAP1 or Cys-202 in HsMetAP1 opens up the S1 pocket. The thiopeptolides Leu-S-Gly-Phe and Phe-S-Gly-Phe, with previously unacceptable Leu or Phe as the N-terminal residue, became efficient substrates of EcMetAP1(C70A) and HsMetAP1(C202A). The relaxed specificity shown in these S1 site mutants for the N-terminal residues was confirmed by hydrolysis of peptide substrates and inhibition by reaction products. The structural features at the enzyme active site will be useful information for designing specific MetAP inhibitors for therapeutic applications.     

Temporal Coherence of Chlorophyll a during a Spring Phytoplankton Bloom in Xiangxi Bay of Three‐Gorges Reservoir,China

Algal bloom phenomenon was defined as “the rapid growth of one or more phytoplankton species which leads to a rapid increase in the biomass of phytoplankton”, yet most estimates of temporal coherence are based on yearly or monthly sampling frequencies and little is known of how synchrony varies among phytoplankton or of the causes of temporal coherence during spring algal bloom. In this study, data of chlorophyll a and related environmental parameters were weekly gathered at 15 sampling sites in Xiangxi Bay of Three‐Gorges Reservoir (TGR, China) to evaluate patterns of temporal coherence for phytoplankton during spring bloom and test if spatial heterogeneity of nutrient and inorganic suspended particles within a single ecosystem influences synchrony of spring phytoplankton dynamics. There is a clear spatial and temporal variation in chlorophyll a across Xiangxi Bay. The degree of temporal coherence for chlorophyll a between pairs of sites located in Xiangxi Bay ranged from –0.367 to 0.952 with mean and median values of 0.349 and 0.321, respectively. Low levels of temporal coherence were often detected among the three stretches of the bay (Down reach, middle reach and upper reach), while high levels of temporal coherence were often found within the same reach of the bay. The relative difference of DIN between pair sites was the strong predictor of temporal coherence for chlorophyll a in down and middle reach of the bay, while the relative difference in Anorganic Suspended Solids was the important factor regulating temporal coherence in middle and upper reach. Contrary to many studies, these results illustrate that, in a small geographic area (a single reservoir bay of approximately 25 km), spatial heterogeneity influence synchrony of phytoplankton dynamics during spring bloom and local processes may override the effects of regional processes or dispersal. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)