Enzymes of cellular metabolism

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Cellular Differentiation of Sucrose Metabolism in Anabaena variabilis 

Cellular Differentiation of Sucrose Metabolism in Anabaena variabilis 

778 N. Schilling and K. Ehrnsperger • Metabolism of Sucrose in Anabaena variabilis O f the sucrose synthesizing enzymes only sucrose synthase was found to be active in extracts from Anabaena variabilis, this activity being ten times higher in vegetative cells than in heterocysts. A sucrose cleaving activity can be attributed to an alkaline invertase (pH optimum 7.5—7.8; data not shown), the activity of which was associated almost exclusively with the heterocysts (19.2 mU). The time course of the solubilization of alkaline invertase during pressure treatment resembles that of glucose- 6-phosphate dehydrogenase.
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Resveratrol inhibits key steps of steroid metabolism in a human estrogen-receptor positive breast cancer model: impact on cellular proliferation

Resveratrol inhibits key steps of steroid metabolism in a human estrogen-receptor positive breast cancer model: impact on cellular proliferation

steroid sulfation by this enzyme can be therefore excluded ( Falany et al., 2002 ; Hruz et al., 2008 ). As the sulfation of E1, E2, and DHEA was strongly inhibited by RES in MCF- 7 cells even upon addition of low concentrations, we also investigated a possible regulation of SULT1A1 and SULT2A1 mRNA expression after incubating MCF-7 cells with 10 µM RES. RES at this concentration showed an almost complete inhibition of E1 and E2 sulfation (91.6 ± 1.1% and 90.0 ± 1.0%, respectively, see Figures 6D, 7A), but had no effect on the cellular proliferation (Figure 1B) even after 48 h. Possible candidates for RES mediating a down-regulation of estrogen sulfating enzymes are the AhR and the pregnane X receptor (PXR) ( Casper et al., 1999 ; Kodama and Negishi, 2013 ; Moscovitz et al., 2018 ). Both receptors are known to regulate mRNA expression of estrogen- metabolizing enzymes including SULTs. Because RES is known to work as an antagonist for AhR and PXR and is capable of down- regulating both nuclear receptors and SULTs, RES might inhibit estrogen sulfation via these pathways.
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Integrating Cellular Metabolism into a Multiscale Whole-Body Model

Integrating Cellular Metabolism into a Multiscale Whole-Body Model

Following the rational of network validation as used in HepatoNet1 [26] we here applied case-specific objectives such as maximization of ammonia production or maximization of uric acid production to quantify extracellular exchange rates with regard to a specific set of boundary conditions. Notably, intracellular flux distributions of biological relevance can hardly be identified using these functional objectives since they rather evaluate the macroscopic behavior of the cell. In contrast, the underlying flux space is assessed qualitatively. In our approach, a compound in the PBPK model can act either as a regulatory modifier or as a substrate of an enzymatic reaction in the metabolic network. We therefore considered two distinct ways of coupling PBPK models and stoichiometric network models: (1) indirect coupling, where concentrations of a compound in the PBPK model impose a regulatory effect on enzyme activity which is quantified at the cellular level (‘feed-forward’), thereby restricting fluxes through this specific reaction and (2) direct coupling, where perturbed metabolic processes (for instance inhibited enzymes) iteratively affect availability of a substance in the PBPK model by directly interfering the corresponding mass balance (‘feed-back’). In both cases, the intracellular concentra- tion of a compound constrains a metabolic state in the underlying network structure [35]. This also influences further downstream events, since the catabolic or anabolic products formed within the intracellular metabolic network are again distributed at the whole organism level. This centralized consideration of metabolism as a core component in human physiology can be seen as an hourglass or bow-tie scheme (Figure 1 A) [36,37]. In particular, enzymatic blockage results in accumulation of the upstream substrate, Author Summary
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Regulation of antioxidant enzymes in Entamoeba histolytica (Schaudinn, 1903)

Regulation of antioxidant enzymes in Entamoeba histolytica (Schaudinn, 1903)

75 Thioredoxin reductase (TrxR), through its immunfluorescence localization, was found to be localized in the cytosol (Figure 3.4). Comparing the two clones, there was no significant difference in the amount of the corresponding proteins between them. This finding suggests the importance of TrxR for both clones, as a redox partner within the TrxR/Trx system, in the metabolism pathway, which could be critical for the maintenance and virulence of the parasite when exposed to highly toxic reactive oxygen species (Arias et al., 2008; Becker et al., 2000). In addition, the thioredoxin redox system is involved in a variety of important cellular functions, which include the maintenance of the intracellular reducing environment, the reduction of ribonucleotides and hence DNA synthesis, and the regulation of transcription of certain genes by interaction with transcription factors (Krnajski et al., 2002). The immune localization of thioredoxin reductase matches the results of RT-PCR, which demonstrated that there is no significant difference in its expression between the two clones A and B.
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Varieties of groups and cellular covers

Varieties of groups and cellular covers

[u, l] ∈ V for all l ∈ L and u ∈ U. This shows that [U, L] ⊆ V and hence [[x, y], y] ∈ V . As B is abelian, also [[x, y], x] = 1 ∈ V holds and [x, y]V ∈ zN follows for N := ⟨xV, yV ⟩. Furthermore, N ′ = ⟨[x, y]V ⟩ N ⊆ zN. Thus we conclude that [N, N ′ ] = 1 and N is a nilpotent subgroup of class 2 of the group W/V . Note that N is finite as ⟨x, y⟩ = ⟨x⟩ wr⟨y⟩ ⊆ W with x and y of finite order. Consequently, N is the cartesian product of its Sylow subgroups (see Theorem 2.1.5). Let N p and N q be the maximal p-subgroup and the
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Elastic interactions of cellular force patterns

Elastic interactions of cellular force patterns

Although the modeling of cell behavior in fibrous gels is beyond the scope of this work our model suggests that at low cell density structure forma- tion is largely independent of the exact material properties (as long as they are isotropic) and one expects alignment of cells into short strings or rings, with- out long-ranged correlation between strings because of the effective screening of elastic signals in the horizontal direction with respect to the string’s axis. For dipoles in 3D positioned on a simple cubic lattice, simulations show that the optimal state exhibits a similar transition between effectively isotropic and aligned structures as a function of Poisson ratio as in 2D. In incompressible substrates (ν = 0.5), we find a hedgehog-like structure, where all dipoles at the corners point to the cube’s center, see Fig. 5.13(a), while for ν = 0 spon- taneous symmetry breaking along a principal lattice lattice vector occurs, see Fig. 5.13(b). For (isotropic) hydrogels typically ν = 0.5, and we therefore do not expect cells to spontaneously align due to elastic interactions in gels with isotropic material properties. However, anisotropic gel properties, e.g. caused by an alignment of collagen fibers, favor cell alignment because the elasticity along the fibers is expected to be larger than in the transverse direction. In this case, cellular traction forces could further stabilize cell alignment by putting fibers under tension. We indeed observe a similar effect in our simulations, when an elastic anisotropy is induced by external strain, see Fig. 5.13(c). The picture shows a snapshot of a Monte Carlo simulations of 100 hard spheres with an elastic dipole moment at their center, where we allowed for both ori- entational and positional degrees of freedom (T ? = 2). In the simulation, we
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Identification of cellular automata: theoretical remarks

Identification of cellular automata: theoretical remarks

• nonlinear: in the quantities mentioned above appear products of variables, or variables raised to the second power or bigger, or any other mathematical operator that lays out the previous definition. Linear systems theory is well developed because the superposition principle holds: a linear system can be divided into parts that are independent and as a consequence can be studied separately, the effects on the whole system being the sum of the single ones. Because of the superposition principle, such problems can often be broken into simpler pieces that can be solved individually, and then the results can be added together. The superposition principle does not hold for nonlinear systems, so many difficulties arise which prevent us from using tools developed in linear theory.
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Dynamics and Structure of Cellular Aggregation

Dynamics and Structure of Cellular Aggregation

Bunde and Havlin (1994) collected a number of examples and application for these con- cepts in natural sciences. In a strict mathematical sense fractals do not have a characteristical length scale. This means if one sees a picture of a fractal where 1 cm represents 10 km and another picture of the same fractal, where 1 cm represents 0.01 cm one can not relate either of the pictures to the used scale. However, in most natural patterns this is only true for all scales but for a good range of scales, because all natural things have a finite maximal size while the smallest (biologically relevant) scale is reached with molecules. Cellular aggregates have a fi- nite size, so for lengths larger than this size the single cell yields a characteristic length-scale. On scales smaller than the size of a single cell the fractal properties are not applicable.
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D-arabitol metabolism of Corynebacterium glutamicum

D-arabitol metabolism of Corynebacterium glutamicum

When focusing on the luciferase activities in the course of growth, it was noticed that luminescence showed specific characteristics in most of the analyzed test strains: after inoculation, luminescence started from a basis level until the mid-exponential growth phase, which was usually after 6 h of cultivation. Maximal luciferase activity seemed to be due to the promoter employed and the carbon source the test strain was grown on. However, all luminescence courses had in common that, after reaching the maximum level, luciferase activity decreased drastically and after 24 h of growth, luminescence levels always were even below the basis level at the beginning. Regarding the decay of luminescence after the 6 h time point, it was remarkable that the half-life of the luciferase usually averaged between 3.5 h and 4.5 h. Thus, the question arose, whether these values displayed the actual half-life of the luciferase in living cells or whether the luciferase protein is subject to active protein degradation mechanisms in C. glutamicum. In order to answer this question, C. glutamicum ∆atlR (pET2_luxABfre_prbtT) was grown in minimal medium containing 1% (w/v) glucose and after 2 h of cultivation, different amounts of spectinomycin were added to the cultures in order to prevent protein biosynthesis. Subsequently, growth and luminescence of the cultures were monitored over a period of 10 h. In Fig. 32A, growth and luciferase activities of cultures with 400 µg and 800 µg spectinomycin/ml are shown, the time point of antibiotic additions is marked as t = 0 h. As a control luminescence and growth of C. glutamicum ∆atlR (pET2_luxABfre_prbtT) without spectinomycin addition is given in Fig. 32B. From the decay of luminescence shown in Fig. 32A, a half-life of around 8 h was calculated for the luciferase protein after shut off of the protein biosynthesis. In contrast, the control culture without antibiotic (Fig. 32B) displayed a distinct faster decrease of luciferase activities after the mid- exponential maximum. This result indicated that there might be an active degradation of proteins after the mid-exponential growth phase in C. glutamicum. An active degradation obviously is prevented by addition of spectinomycin as synthesis of proteases is blocked by
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Co-expression of cellulolytic enzymes in plant cells

Co-expression of cellulolytic enzymes in plant cells

In case of E2, expression in E. coli was reported for crystal structure determination (Larsson et al., 2005). Expression of E3 was performed for characterization of the enzyme rather than enzyme production, as well (see above and Vuong and Wilson, 2009a; Vuong and Wilson, 2009b). For E4, in addition to reports where the E. coli expressed enzyme was used for protein characterization without statement of specific yields (Escovar-Kousen et al., 2004; Kostylev et al., 2012; Li et al., 2007b), one publication described yields of purified protein in the range of 10-25 mg/l (Zhou et al., 2004). Endoglucanase E5 was expressed in several experiments in E. coli, but only a recent study reports activities of approx. 18 U/ml*h on CMC for fermentation at lab scale (Yan et al., 2013). For exoglucanase E6, expression was performed without declaration of yields for protein characterization and implementation into cellulosomes (Caspi et al., 2008; Kostylev et al., 2014; Kostylev and Wilson, 2013; Kostylev and Wilson, 2011; Moraïs et al., 2010). Endoglucanase E1 is the only protein which has not been expressed successfully in E. coli to this day. Although the cellulases from T. fusca have been produced in E. coli before, most of the experiments were focused on fundamental studies of the enzymes rather than on production of high amounts of protein for cellulose degradation. This was solely reported for E5 by Yan and coworkers (2013). Furthermore, the examples mentioned above employed IPTG for expression of T. fusca cellulases. Thus, the constitutive system employing the prrn promoter described here could be interesting for further studies to estimate possible yields and activities and eventually provide an alternative route for cost efficient cellulase expression in E. coli at lower enzyme production expenses. This is in particular interesting for the expression of BglC, since low amounts of β-glucosidases are a bottleneck for more efficient cellulase mixtures (Juhász et al., 2005; Zhou et al., 2009a).
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Analysis of the sliding process of restriction enzymes

Analysis of the sliding process of restriction enzymes

The conformational changes could best be explained assuming a twisting-like motion of the two subunits of BsoBI, by which upon specific binding the DNA-binding cleft closes while the residues located at position 153 however move away from each other [105]. If with this explanation, the DNA-binding cleft is considered to be closed in the specific complex and open in the apoenzyme, this would consequently mean that the DNA-bind- ing cleft is also open in the non-specific complex, since the FRET efficiencies of the apoenzyme and the non-specific complex were found to be similar (0.46 vs. 0.45). No change of the FRET efficiency upon binding to a non-specific 12 bp oligonucleotide has been observed also by Jasmina Dikic in single-molecule experiments [105]. Interestingly, the addition of a non-specific 39 bp oligonucleotide induced a decrease of the FRET ef- ficiency, which in a twisting-like motion would mean a closing of the DNA-binding cleft. It was therefore concluded that the conformation BsoBI uses for sliding is more closed than the conformation of the apoenzyme [105]. It can be seen that the conformational changes of BsoBI are more complex and cannot be explained by single FRET measure- ments. A definitive answer regarding the non-specific complex of BsoBI requires further experiments (e.g. labeling at different sites in the protein) or the solution of the crystal structure. Nevertheless, it is not assumed that BsoBI greatly deviates from its ‘tunnel conformation’, because a tunnel could be a strategy to promote linear diffusion [36]. However, it is assumed that the conformation BsoBI adopts for sliding is ‘tunnel-like’, on the one hand closed enough to prevent falling off the DNA and on the other hand open enough to prevent friction between the protein and the DNA.
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Molecular dynamics simulations of enzymes on surfaces

Molecular dynamics simulations of enzymes on surfaces

resolved loop region (residues 85-95) and all hydrogens were added with the CHARMM package. 16 Amino acid side chains and histidine residues were protonated according to pH 7 in their environment. In particular, the heme-coordinating histidines were protonated at the δ-nitrogen atoms following the model proposed by Leu et al. 17 This procedure gives a strongly negatively charged protein model with a net charge of -36 e. Afterward, the system was solvated in a rectangular box (174  119  120.5 Å 3 ) with explicit TIP3P water molecules 18 and neutralized by the addition of sodium ions with VMD1.8.6. 19 The all-atom simulations were performed with NAMD2.6 20 using the CHARMM32b force field. 21 For the oxidized heme cofactors and the two ligating histidines, we used the parameter set and the partial charges derived by Leu et al. 17 The molybdopterin including the Mo atom was treated as a rigid body using free energy calculations to restrain all internal coordinates. Nonbonding parameters for the Moco, derived by Metz et al. with DFT methods, were applied. 22 After energy minimization, heating, and equilibration of the system of ∼236.000 atoms, an SMD was performed for 1 ns, pulling the cytochrome b5 domain of chain A sequentially toward the Moco cavity. For monomer B, no SMD was applied, but we involved it in the MD simulations to maintain the interaction area between the two monomers. To retain the secondary structure of the cytochrome b5 domain during the presence of artificial pulling forces, we applied light restraints on its backbone torsions. Additionally, all backbone atoms located outside a 10 Å sphere from the heme iron were restrained to their positions by weak forces of 0.01 kcal/ mol 3 Å 2 . To check for the stability of the gained conformation, a 14 ns long MD relaxation and equilibration phase followed the SMD. All calculations were carried out in an NPTensemble at 300 K and atmosphere pressure using the Langevin piston method 23 with a time step of 2 fs enabled by rigid bonds applied to all hydrogens. 24 The simulations were performed with periodic boundary conditions in all direc- tions, the particle-mesh-Ewald summation for electrostatic interactions, 25 and a simple cutoff of 12 Å for van der Waals interactions.
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Enzymes of two clostridial amino-acid fermentation pathways

Enzymes of two clostridial amino-acid fermentation pathways

NADH is the electron carrier providing the reducing equivalents in the reductive branch of the process and reduced ferredoxin must be reoxidised in order to maintain the fermentation equilibrium. These facts supported the suggestion on the existence of a NADH:ferredoxin oxidoreductase in C. tetanomorphum that may conserve energy in form of an electrochemical gradient. In this sense, at beginning of this work, two proteins were investigated to be possibly involved with a membrane dependent NADH activity: butyryl-CoA dehydrogenase and a novel NADH dehydrogenase. The latter enzyme could be successfully purified and was identified as an Rnf-type protein in membrane extracts of C. tetanomorphum. The Rnf complex originally described in Rhodobacter capsulatus is proposed to function as an oxidoreductase that reduces ferredoxin at the expense of the membrane potential generated from electron donors like NADH (Kumagai et al, 1997; Jouanneau et al, 1998; Boiangiu et al, 2005). Nevertheless, butyryl-CoA dehydrogenase (crotonyl-CoA reductase) had also been suggested to participate in energy conservation, similar to anaerobic respiration, based on the high redox potential difference observed between the redox couple crotonyl-CoA/butyryl- CoA (E 0 ' = -10 mV) and the NAD + /NADH (E 0 ' = -320 mV) pair. The enzyme described in this work was purified under oxic conditions following only the oxidation of butyryl-CoA with ferricenium hexafluorophosphate, which is an electron acceptor generally used to screen acyl-CoA dehydrogenase activity (Lehman & Thorpe, 1990). The N-terminal sequence determination and molecular composition of the native enzyme shows a great similarity of the enzyme to the propionyl-CoA dehydrogenase isolated from C. propionicum (Hetzel et al, 2003). Both enzymes have the same subunit composition: α-subunit (propionyl-CoA or butyryl-CoA dehydrogenase), a large ETF β-subunit (36 kDa) and a small ETF γ-subunit (28 kDa), in a possible α 2 βγ structure. A closely related enzyme of butyryl-CoA dehydrogenase
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Enzymes for the synthesis of bioactive compounds

Enzymes for the synthesis of bioactive compounds

Results and discussion 3.1.5 Discussion Applying a heterologous three-component system (CYP107D1+PdR/PdX) in the bioconversion of LCA, we have shown that CYP107D1 is not only involved in oleandomycin biosynthesis as described by Montemiglio et al., but also exhibits excellent regio- and stereoselectivity for LCA and DCA, producing 6β-hydroxylated bile acids exclusively (Montemiglio et al. 2015; Parisi et al. 2019). Agematu et al. expressed a CYP107D1 with PdR/PdX as a redox partner in E. coli and reported OleP hydroxylated testosterone at the 6β-, 7β-,12β and 15β- positions, indicating that OleP was a multifunctional P450 (Agematu et al. 2006). The different selectivity of OleP, observed in this study, can be explained by the differences in the steroidal structure. While testosterone contains a double bond between C4 and C5 and the A/B ring fusion has a trans conformation, the A/B ring in the bile acids is cis, creating a significant spatial difference between the molecules (Li and Chiang 2014). Hydroxylation of LCA in 6β-position was also described in the literature for eukaryotic P450 monooxygenase from Golden hamster CYP3A10, which has only 20% similarity to OleP (Teixeiras and Gilg 1991; Chang et al. 1993). In humans, by contrast, P450 CYP3A4 hydroxylate steroid hormones on position 6β can also metabolize lithocholic acid, but in this case the bile acid is hydroxylated at the 6α position. Since DCA is widely used in medicine and the possibility of producing its metabolites has become important. The synthesis of 6β- hydroxylated DCA starting from DCA was previously described in 11 steps with an overall yield below 20% which is time-consuming and a complicated procedure compared to one-step biotransformation using CYP107D1 (Iida et al. 1986). OleP is the first bacterial P450, which selectively hydroxylates bile acids on the 6β position and thus provides the opportunity to use it as an alternative drug target for metabolic disorders, cancer, and also as antimicrobial agent. In summary, CYP107D1 from Streptomyces antibioticus represents a promising new biocatalyst for hydroxylation reactions. The enzyme can be easily expressed with the redox partners PdX/PdR in E. coli. OleP has been found to hydroxylate certain bile acids in a regio- and stereoselective manner. The multifunctionality of OleP opens up new horizons for the commercial production of physiologically active steroids, which are difficult to obtain from alternative steroid raw materials.
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Anzeige von Enzymes in everyday life

Anzeige von Enzymes in everyday life

Für den Fall, dass Schulen nicht gewillt sind, die enormen Umstrukturierungen vorzunehmen, die erforderlich werden, wenn ein bilingualer Zug etabliert werden soll, bietet sich der Eins[r]

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Investigations into Enzymes of Nitrogen Metabolism of the Ectomycorrhizal Basidiomycete, Suillus bovinus Fax: 0 5 2 1 -1 0 6 -6 0 3 9 . E-mail 

Investigations into Enzymes of Nitrogen Metabolism of the Ectomycorrhizal Basidiomycete, Suillus bovinus Fax: 0 5 2 1 -1 0 6 -6 0 3 9 . E-mail 

210 N. Grotjohann et al. ■ Enzymes o f N itrogen M etabolism of S u illus b o vin u s The strict dependence on N A D H /N A D + of glu­ tam ate dehydrogenase of our Suillus bovinus iso­ lates is most rem arkable different from respective reports in literature which describe either NADH- plus N A D PH -dependent or only NAD PH -depen- dent glutam ate dehydrogenases for various mycor- rhizal fungi ( Sphaerostilbe repens, Botton and Msatef, 1983; Cenococcum graniforme, Martin et al., 1983, 1988; Laccaria bicolor , Ahm ad et al., 1990; Chalot et al., 1991; Stropharia sem iglobata , Schwartz et al., 1991). Rudawska et al. (1994) re­ port the N A D PH -dependent enzyme species even for Suillus bovinus. Using isolates from Poland, this discrepancy can only be taken as another ex­ ample of the repeatedly discussed metabolic dif­ ferences in various isolates of presumably identical fungal species.
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Targeted quantification of central metabolic enzymes in Corynebacterium glutamicum

Targeted quantification of central metabolic enzymes in Corynebacterium glutamicum

Figure 5.16: Synthesis, cloning and expression of QConCat protein standards for absolute quantification of central metabolic enzymes in C. glutamicum. A: Plasmid maps of the two expression vectors carrying concatenated proteotypic peptides for glycolysis and anaplerosis related enzymes (pBAD::EA QCC ) as well as TCA-cycle and glyoxylate shunt enzymes (pBAD::TG QCC ). Genes are depicted as arrows, regulatory sequences as boxes. Commonly used single cutter restriction endonuclease sites are displayed. B: Gel electrophoretic analysis of restriction fragments of QConCat expression plasmids isolated from E. coli strains BL21 (DE3) and TOP10. Plasmids were isolated using Qiagen plasmid Mini-Kit from overnight cultures in LB medium with 50 µg ml -1 ampiciline, cut with XhoI and PstI at 37 °C for 1 h and finally separated in a 1 % agarose-TEA gel. M: Fermentas 1kb DNA ladder. C: Immunological detection of Strep- tagged QConCat protein standards. QConCat expression was induced in E. coli protein expression strains BL21 (DE3) and TOP10 during growth on LB media with 0.2 % (w v -1 ) L-arabinose. Crude extracts were analyzed by SDS-PAGE. C-terminal Strep tag epitopes on target proteins were detected after Western blotting by antibody coupled immunostaining (anti Strep: Qiagen 34850; secondary antibody: anti mouse IgG AP coupled Sigma Aldrich A3562, staining with NBT/ BCIP).
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Studies to influence the carbon metabolism of the C 3 plant Nicotiana tabacum by expressing heterologous enzymes involved in C 4 -like CO 2 fixation

Studies to influence the carbon metabolism of the C 3 plant Nicotiana tabacum by expressing heterologous enzymes involved in C 4 -like CO 2 fixation

types in combination. One plant (JL18) showed a trend of decrease in the e/A ratio, although this effect was observed only in two of five measurements (4.5.4.3). In addition, some other plants exhibited increases in A only at some time points. It seems likely that in these plants the C4-metabolic pathway(s) operated only transiently for a short-term under certain physiological conditions. This reminds us of some C 3-C4 Flaveria species which showed some C4-like CO2 fixation during short-term study on the carbon isotope discrimination, however, exhibited C3-like values in long-term measurements (review by Rawsthorne, 1992). In the case of transgenic tobacco plants, the extent to which this short-term C 4-metabolism operates might be depending on several factors. 1) CO 2 leakage: C 3 plants have no Kranz anatomy that is responsible for preventing leakage of CO2 in C4 plants (1.2). CO2 generated by the decarboxylation of OAA and/or malate in chloroplasts could again be released instead of being concentrated there. Although one terrestrial plant, B. aralocaspica, that lacks Kranz anatomy employs C 4-type photosynthesis, it has a special leaf anatomy. Rubisco and the enzymes involved in C4-metabolism (e.g. NAD-ME and DK) are localized in different types of chloroplasts, which are present in basal and distal parts of their chlorenchyma cells, respectively (Freitag and Stichler, 2000; Vozesenskaya et al., 2001). 2) the availability of substrates and co- factors. 3) post-translational regulation of PC and DK (for review see Leegood, 1997). 4) the cooperation of individual enzymes in vivo: the physiological conditions could influence the enzyme activities, for instance through changing the activation states of foreign plant-derived unmodified PC, DK and CK, and of endogenous NADP-MDH. It is thus possible that under certain physiological conditions in tobacco plant JL18 and some other plants, the rates of reactions catalysed by individual “C4-cycle” enzymes matched each other, which resulted in short-term effects. However, when the physiological conditions shifted far from this “point”, one or more reaction(s) became rate- limiting, “switching-off” C4-like metabolism. It might be that in these transgenic plants the changes in their metabolism caused by constitutive expression of the “C4-cycle” genes were so large that the plants frequently had to adjust their intracellular conditions to maintain their metabolic balances.
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Chloroplast RNA metabolism

Chloroplast RNA metabolism

Ribonucleases have long been considered as purely degradative enzymes, cleaving without any sequence specificity. But nowadays a greater awareness of the significance of both endo- and exoribonucleases is emerging, in particular because it has become apparent that many RNases can display specificity for sequences or structures, allowing them to regulate transcript abundance according to the needs of the cell. It is becoming obvious that RNases play a central role in RNA metabolism, including RNA decay, maturation of RNA precursors and end-trimming of certain RNAs. A contribution of RNases to the implementation of molecular processes in response to environmental signals is emphasizing the importance of RNA metabolism in adjusting chloroplast functions. A single cell or organelle contains various RNases with sometimes overlapping functions and specificities, and high-molecular-weight complexes function together with stabilizing elements in order to control overall RNA accumulation (Stoppel and Meurer, 2011). Besides the protection of RNA termini by RNA secondary structures, RNA stability is influenced by polyadenylation or ribosome binding and in vivo RNA cleavage specificity is
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Manganese metabolism in humans

Manganese metabolism in humans

While great strides have been made in the past years, the complete picture of Mn metabolism and homeostasis remains to be mapped out. Advanced biomarkers or measuring techniques are needed to monitor body Mn levels, especially for chronic Mn accumulation. As blood Mn is very dynamic and only represents current body Mn levels in a relatively short term, patients with low blood Mn after chelation therapy might still suffer from Mn-induced neurotoxicity due to slow release of Mn from other tissues accumulated in the past. Bone Mn is a good biomarker for this purpose, given that bone stores the largest amount of Mn in human body and bone Mn has a long half-life (skeleton bone 8.5. years). Recent techniques such as in vivo neutron activation analysis (IVNAA) allow non-invasive measurement of bone Mn levels, thus are of great help to access chronic Mn accumulation and monitor Mn levels across developmental stages in children. Brain is the primary target of Mn poisoning. As for regulation of Mn homeostasis, although we have identified a few transporters capable to transport Mn, a large numbers of regulator proteins remain to be identify. Besides, among these known transporters, actually none of them has been proved as a Mn specific transporter. Most of them facilitate couple metal ions influx or efflux with highest affinity to other metals rather than Mn. SLC30A10 could be a potential candidate, but more research is needed to confirm that. In addition, within a cell, the nucleus is the largest Mn storage site with the highest Mn concentration. However, regulation of Mn homeostasis in this organelle remain blank due to very few intracellular transporters identified there. A forward or reverse genetic screen to find these transporters or regulators is of great interest and priority to understand Mn homeostasis in cells and in human body.
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