Synechococcus is one of the most important contributors to global primary productivity, and ocean warming is predicted to increase abundance and distribution of Synechococcus in the ocean. Here, we investigated molecular response of an oceanic Synechococcus strain WH8102 grown in two nitrogen sources (nitrate and urea) under present (25°C) and predicted future (28°C) temperature conditions using an isobaric tag (IBT)-based quantitative proteomic approach. Rising temperature decreased growth rate, contents of chlorophyll a, protein and sugar in the nitrate-grown cells, but only decreased protein content and significantly increased zeaxanthin content of the urea-grown cells. Expressions of CsoS2 protein involved in carboxysome formation and ribosomal subunits in both nitrate- and urea-grown cells were significantly decreased in rising temperature, whereas carbohydrate selective porin and sucrose-phosphate synthase (SPS) were remarkably up-regulated, and carbohydrate degradation associated proteins, i.e., glycogen phosphorylase kinase, fructokinase and glucose-6-phosphate dehydrogenase, were down-regulated in the urea-grown cells. Rising temperature also increased expressions of three redox-sensitive enzymes (peroxiredoxin, thioredoxin, and CP12) in both nitrate- and urea-grown cells. Our results indicated that rising temperature did not enhance cell growth of Synechococcus; on the contrary, it impaired cell functions, and this might influence cell abundance and distribution of Synechococcus in a future ocean.

Thioredoxin reductases control the redox state of thioredoxins (Trxs)-ubiquitous proteins that regulate a spectrum of enzymes by dithiol-disulfide exchange reactions. In most organisms, Trx is reduced by NADPH via a thioredoxin reductase flavoenzyme (NTR), but in oxygenic photosynthetic organisms, this function can also be performed by an iron-sulfur ferredoxin (Fdx)-dependent thioredoxin reductase (FTR) that links light to metabolic regulation. We have recently found that some cyanobacteria, such as the thylakoid-less Gloeobacter and the ocean-dwelling green oxyphotobacterium Prochlorococcus, lack NTR and FTR but contain a thioredoxin reductase flavoenzyme (formerly tentatively called deeply-rooted thioredoxin reductase or DTR), whose electron donor remained undefined. Here we demonstrate that Fdx functions in this capacity and report the crystallographic structure of the transient complex between the plant-type Fdx1 and the thioredoxin reductase flavoenzyme from Gloeobacter violaceus. Thereby, our data demonstrate that this cyanobacterial enzyme belongs to the Fdx flavin-thioredoxin reductase (FFTR) family, originally described in the anaerobic bacterium Clostridium pasteurianum. Accordingly, the enzyme hitherto termed DTR is renamed FFTR. Our experiments further show that the redox sensitive peptide CP12 is modulated in vitro by the FFTR/Trx system, demonstrating that FFTR functionally substitutes for FTR in light-linked enzyme regulation in Gloeobacter. Altogether, we demonstrate the FFTR is spread within the cyanobacteria phylum and propose that, by substituting for FTR, it connects the reduction of target proteins to photosynthesis. Besides, the results indicate that FFTR acquisition constitutes a mechanism of evolutionary adaptation in marine phytoplankton such as Prochlorococcus that live in low-iron environments.

Keywords: CP12; FFTR; NTR; cyanobacteria; disulfide; ferredoxin; photosynthesis; thioredoxin.

The parent compound 1,3-dithiane (L1) was reacted with CuI providing the 1D coordination polymer [{Cu(μ₂-I)₂Cu}(μ₂-L1)₂] _n (CP1), an isostructural compound [{Cu(μ₂-Br)₂Cu}(μ₂-L1)₂] _n (CP2) was isolated upon treatment of CuBr with L1. In contrast, treatment of L1 with CuCl results in the formation of 2D polymeric [{Cu(μ₂-Cl)₂Cu}(μ₂-L1)] _n (CP3), in which each sulfur atom acts as a 4-electron donor. The 1D compounds [{Cu(μ₂-X)₂Cu}(μ₂-L2)₂] _n (CP7, X = Br, and CP8, X = Cl) resulting from treatment of 2-methyl-1,3 dithiane (L2) with CuBr and CuCl are isostructural with their CuI homologue [{Cu(μ₂-I)₂Cu}(μ₂-L2)₂] _n (CP5), reported previously. Using CuCN, a 2D CP of composition [{Cu(μ₂-CN)₂Cu}(μ₂-L2)₂] _n (CP9) has been isolated. Complexation of 2-isobutyl-1,3-dithiane (L3) on CuI generates a 2D material [{Cu₃(μ₃-I)(μ₂-I)₂(μ₂-L3)₂}] _n (CP10), incorporating the usual trinuclear μ₃-I-capped Cu clusters as SBUs, whereas 2D-polymeric compounds [{Cu(μ₂-Br)₂Cu}(μ₂-L3)₂] _n (CP11) and [{Cu(μ₂-Cl)₂Cu}(μ₂-L3)₂] _n (CP12) were obtained with CuBr and CuCl. Treatment of 2-Me₃Si-1,3-dithiane (L4) with CuX yields the series [{Cu₂(μ₄-X)(μ₂-X)}(μ₂-L4)] _n (CP13-CP15). With 2-phenyl-1,3-dithiane (L5), the outcome of the reaction with CuI depends on the reaction conditions. Reaction with CuI in MeCN provides a 1D ribbon [{Cu(μ₂-I)₂Cu}(MeCN)₂(μ₂-L5)₂] _n (CP16), whereas treatment of CuI with L5 in hot EtCN yields 2D-polymeric[{Cu₃(μ₃-I)(μ₂-I)₂(μ₂-L5)₂}] _n (CP17). A reversible phase transition from triclinic P1̅ to monoclinic P2₁/ m is observed when recording the structure of CP16 at five different temperatures in the 100-300 K range. Ligand L6 containing a ferrocenyl function at the 2-position was also probed as organometallic dithioether ligand. Reaction of L6 with 1 equiv of CuI produces the 0D dinuclear complex [{Cu(μ₂-I)₂Cu}(η¹-L6)₂(MeCN)₂] (D1), whereas treatment with 2 equiv of CuI affords the novel 1D CP [{Cu(μ₃-I)₂Cu}(μ-L6)] _n (CP18), in which both S atoms of one L6 molecule span two copper centers of the infinite (CuI) _n ribbon. Some selected results of thermal analyses and luminescence measurements are also presented.

With the objective to establish a correlation between the spacer distance and halide dependence on the structural features of coordination polymers (CPs) assembled by the reaction between CuX salts (X = Cl, Br, I) and dithioether ligands BzS(CH₂)_nSBz (<i>n</i> = 1-9; Bz = benzyl), a series of 26 compounds have been prepared and structurally investigated. A particular attention has been devoted to the design of networks with extremely long and flexible methylene spacer units between the SBz donor sites. Under identical conditions, CuI and CuBr react with BzSCH₂Bz (<b>L1</b>) affording respectively the one-dimensional (1D) CPs {Cu(μ₂-I)₂Cu}(μ-<b>L1</b>)₂]<i>n</i> (<b>CP1</b>) and {Cu(μ₂-Br)₂Cu}(μ-<b>L1</b>)₂] (<b>CP2</b>), which incorporate Cu(μ₂-X)₂Cu rhomboids as secondary building units (SBUs). The hitherto unknown architecture of two-dimensional (2D) layers obtained with CuCl (<b>CP3</b>) differs from that of <b>CP1</b> and <b>CP2</b>, which bear inorganic -Cl-Cu-Cl-Cu-Cl- chains interconnected through bridging <b>L1</b> ligands, thus forming a 2D architecture. The crystallographic characterization of a 1D CP obtained by reacting CuI with 1,3-bis(benzylthio)propane (<b>L2</b>) reveals that [{Cu(μ₂-I)₂Cu}(μ-<b>L2</b>)₂]<i>n</i> (<b>CP4</b>) contains conventional Cu₂I₂ rhomboids as SBUs. In contrast, unusual isostructural CPs [{Cu(μ₂-X)}(μ₂-<b>L2</b>)]<i>n</i> (<b>CP5</b>) and (<b>CP6</b>) are obtained with CuX when X = Br and Cl, respectively, in which the isolated Cu atoms are bridged by a single μ₂-Br or μ₂-Cl ion giving rise to infinite [Cu(μ₂-X)Cu]<i>n</i> ribbons. The crystal structure of the strongly luminescent three-dimensional (3D) polymer [{Cu₄(μ₃-I)₃(μ₄-I)(μ-<b>L3</b>)_1.5]<i>n</i> (<b>CP7</b>) issued from reacting 2 equiv of CuI with BzS(CH₂)₄SBz (<b>L3</b>) has been redetermined. <b>CP7</b> features unusual [(Cu₄I₃)(μ₄-I)]<i>n</i> arrays securing the 3D connectivity. In contrast, mixing CuI with an excess of <b>L3</b> provides the nonemissive material [{Cu(μ₂-I)₂Cu}(μ-<b>L3</b>)₂]<i>n</i> (<b>CP8</b>). Treatment of CuBr and CuCl with <b>L3</b> leads to [{Cu(μ₂-Br)₂Cu}(μ-<b>L3</b>)₂]<i>n</i> (<b>CP9</b>) and the 0D complex [{Cu(μ₂-Cl)₂Cu}(μ-<b>L3</b>)₂] (<b>D1</b>), respectively. The crystallographic particularity for <b>CP9</b> is the coexistence of two topological isomers within the unit cell. The first one, <b>CP9</b>-<b>1D</b>, consists of simple 1D ribbons running along the <i>a</i> axis of the unit cell. The second topological isomer, <b>CP9</b>-<b>2D</b>, also consists of [Cu(μ₂-Br)₂Cu] SBUs, but these are interconnected in a 2D manner forming 2D sheets placed perpendicular to the 1D ribbons. Four 2D CPs, namely, [{Cu₄(μ₃-I)₄}(μ-<b>L4</b>)₂]<i>n</i> (<b>CP10</b>), [{Cu(μ₂-I)₂Cu}(μ-<b>L4</b>)₂]<i>n</i> (<b>CP11</b>), [{Cu(μ₂-Br)₂Cu}(μ-<b>L4</b>)₂]<i>n</i> (<b><em>CP12</em></b>), and [{Cu(μ₂-Cl)₂Cu}(μ-<b>L4</b>)₂]<i>n</i> (<b>CP13</b>), stem from the self-assembly process of CuX with BzS(CH₂)₆SBz (<b>L4</b>). A similar series of 2D materials comprising [{Cu₄(μ₃-I)₄}(μ-<b>L5</b>)₂]<i>n</i> (<b>CP14</b>), [{Cu(μ₂-I)₂Cu}(μ-<b>L5</b>)₂]<i>n</i> (<b>CP15</b>), [{Cu(μ₂-Br)₂Cu}(μ-<b>L5</b>)₂]<i>n</i> (<b>CP16</b>), and [{Cu(μ₂-Cl)₂Cu}(μ-<b>L5</b>)₂]<i>n</i> (<b>CP17</b>) result from the coordination of BzS(CH₂)₇SBz (<b>L5</b>) on CuX. Ligation of CuX with the long-chain ligand BzS(CH₂)₈SBz (<b>L6</b>) allows for the X-ray characterization of the luminescent 2D [{Cu₄(μ₃-I)₄}(μ-<b>L6</b>)₂]<i>n</i> (<b>CP18</b>) and the isostructural 1D series [{Cu(μ₂-X)₂Cu}(μ-<b>L6</b>)₂]<i>n</i><b>CP19 (</b>X = I), <b>CP20</b> (X = Br) and <b>CP21</b>(X = Cl). Noteworthy, BzS(CH₂)₉SBz (<b>L7</b>) bearing a very flexible nine-atom chain generated the crystalline materials 2D [{Cu₄(μ₃-I)₄}(μ-<b>L7</b>)₂]<i>n</i> (<b>CP22</b>) and the isostructural 1D series [{Cu(μ₂-X)₂Cu}(μ-<b>L6</b>)₂]<i>n</i><b>CP23</b> (X = I), <b>CP24</b> (X = Br), and <b>CP25</b> (X = Cl), featuring nanometric separations between the cubane- or rhomboid-SBUs. This comparative study reveals that the outcome of the reaction of CuX with the shorter ligands BzS(CH₂)_nSBz (<i>n</i> = 1-4) is not predictable. However, with more flexible spacer chains BzS(CH₂)_nSBz (<i>n</i> = 6-9), a clear structural pattern can be established. Using a 1:1 CuX-to-ligand ratio, [{Cu(μ₂-X)₂Cu}(μ-<b>L4-7</b>)₂] CPs are always formed, irrespectively of <b>L4</b>-<b>L7</b>. Employing a 2:1 CuX-to-ligand ratio, only CuI is able to form networks incorporating Cu₄(μ₃-I)₄ clusters as SBUs. All attempts to construct polynuclear cluster using CuBr and CuCl failed. The materials have been furthermore analyzed by powder X-ray diffraction, Raman spectroscopy, and thermogravimetric analysis, and the photophysical properties of the emissive materials have been studied.

To clarify the regulatory mechanisms of the Calvin cycle in algae, we analyzed the molecular properties of the enzymes involved in this cycle. We demonstrated that these enzymes were not regulated by redox modulation through the ferredoxin/thioredoxin system under light/dark conditions and were not sensitive to treatments with hydrogen peroxide in vitro, unlike the chloroplastic thiol-modulated enzymes of plants. On the other hand, we found that cyanobacteria possessed a unique enzyme involved in the Calvin cycle. The CP12 protein played an important role in regulating carbon metabolism in the Calvin cycle in cyanobacteria and eukaryotic algae. This review described the regulatory mechanisms of the Calvin cycle in algae and also the effects of alterations to photosynthetic carbon metabolism on plant productivity, carbon partitioning, and the carbon/nitrogen balance using transgenic plants expressing algal genes.

Oxygenic phototrophs use the Calvin-Benson cycle to fix CO₂ during photosynthesis. In the dark, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), two enzymes of the Calvin-Benson cycle, form an inactive complex with the regulatory protein CP12, mainly under the control of thioredoxins and pyridine nucleotides. In the light, complex dissociation allows GAPDH and PRK reactivation. The GAPDH/CP12/PRK complex is conserved from cyanobacteria to angiosperms and coexists in land plants with an autoassembling GAPDH that is analogously regulated. With the recently described 3D structures of PRK and GAPDH/CP12/PRK, the structural proteome of this ubiquitous regulatory system has been completed. This outcome opens a new avenue for understanding the regulatory potential of photosynthetic carbon fixation by laying the foundation for its knowledge-based manipulation.

Keywords: metabolism; photosynthesis; protein complexes; redox regulation.

Cinta Senese is characterized by slow growth rates, which implies different nutritional requirements compared to major pig breeds. Four different crude protein levels (120, 140, 160 and 180 g/kg on as-fed basis, denoted as CP12, CP14, CP16 and CP18) were tested to assess the optimal protein requirements of Cinta Senese pigs during the growing phase. The in vivo performance, slaughtering traits and nitrogen balance were evaluated using individual pens and metabolic crates. Increasing the protein level in feed lowered the average daily gain (from 0.76 to 0.71 kg/d), final weight (63.0 kg for CP12 versus 60.7 kg for CP16) and reduced the protein conversion efficiency (from 0.37 to 0.58). Also, protein conversion in lean protein linearly increased from CP12 (4.82) to CP18 (7.43), which implies a worsening in the protein utilization efficiency. The nitrogen balance showed higher loss of N through urine (from 0.68 g/d/kg metabolic weight for CP12 to 1.14 g/d/kg metabolic weight for CP18) as the dietary CP levels increased, and a decrease in the biological value (51.78 for CP12 versus 36.54 for CP16). The results indicated that the CP12 diet was adequate for fulfilling the Cinta Senese protein requirements during the growing phase.

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges is simultaneous. In oxidized CP12, the C₂₃-C₃₁ pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C₆₆-C₇₅ pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C₆₆-C₇₅ pair than for the C₂₃-C₃₁ pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12.

Keywords: Calvin–Benson–Bassham cycle; conditionally disordered protein; intrinsically disordered protein; photosynthesis regulation.

We determined the effects of an immediately antecedent viral lower respiratory tract infection (LRI) on the severity of clinical illness, changes in lung function and airway histamine responsiveness produced by a subsequent LRI in 9-12 week old beagle puppies inoculated with canine adenovirus 2, followed in 2 weeks by inoculation with canine parainfluenza 2 virus (CAV2-CP12, n = 7). We compared their acute responses to puppies infected with CP12 alone (n = 5), CAV2 alone (n = 7), and no infection (control, n = 6). Puppies inoculated with either virus alone developed a LRI 3 to 6 days after inoculation which resolved by 12-14 days after inoculation. However, the illness was more severe in the CAV2 group. In the CAV2-CP12 group, CP12 infection following CAV2 infection resulted in a clinical illness nearly comparable to that observed with CAV2 alone. Whereas in control and CP12 puppies, lung resistance (RL) decreased and dynamic lung compliance (Cdyn) increased during the study due to normal growth, RL increased and Cdyn remained unchanged in the CAV2 group. In contrast, RL did not change and Cdyn increased in the CAV2-CP12 group. Airway histamine responsiveness in the CAV2-CP12 group increased during infection with CP12 and was similar to that observed with CAV2 alone. In contrast, infection with CP12 alone produced a small, but non-significant increase in histamine responsiveness. The duration of the increase in histamine responsiveness was not prolonged in the CAV2-CP12 group in comparison to CP12 or CAV2 alone. However, the length of clinical illness was extended in the CAV2-CP12 group in comparison to the other infected groups. These data suggest that an immediately antecedent viral LRI can potentiate the clinical and physiologic effects of a subsequent viral LRI.

The permeability-controllable potentiometric fluorescent probes that can visually discriminate near-zero and normal situations of cell membrane potential were reported for the first time. Different from traditional potentiometric probes that utilize fluorescence intensity to reflect membrane potential, CQ12 and CP12 have different localizations under the two situations of cell membrane potential. Thus, the two situations can be point-to-point indicated by two fluorescent images with an obvious difference, avoiding complex operations and calibration of conventional methods.

Over the past few decades, bioengineered cyanobacteria have become a major focus of research for the production of energy carriers and high value chemical compounds. Besides improvements in cultivation routines and reactor technology, the integral understanding of the regulation of metabolic fluxes is the key to designing production strains that are able to compete with established industrial processes. In cyanobacteria, many enzymes and metabolic pathways are regulated differently compared to other bacteria. For instance, while glutamine synthetase in proteobacteria is mainly regulated by covalent enzyme modifications, the same enzyme in cyanobacteria is controlled by the interaction with unique small proteins. Other prominent examples, such as the small protein CP12 which controls the Calvin-Benson cycle, indicate that the regulation of enzymes and/or pathways via the attachment of small proteins might be a widespread mechanism in cyanobacteria. Accordingly, this review highlights the diverse role of small proteins in the control of cyanobacterial metabolism, focusing on well-studied examples as well as those most recently described. Moreover, it will discuss their potential to implement metabolic engineering strategies in order to make cyanobacteria more definable for biotechnological applications.

Keywords: biotechnology; metabolic regulation; small proteins.

In this article, one of the grating agencies requested us to incorporate the information, Spanish Government and co-funded by European Union ERDF/ESF, "Investing in your future", in the acknowledgments section. The correct acknowledgement is as follows: "This work has been supported by grants of the Spanish Ministry of Health (Fondo de Investigaciones Sanitarias, PI13/00029, Spanish Government and co-funded by European Union ERDF/ESF, "Investing in your future"), Department of Education of Castilla and Leon Regional Government (Grant# LE093U13) and Mutua Madrileña Foundation (Basic research grants 2012) to J.I.R.B.; by Miguel Servet National Program (Ministry of National Health) CP12/03063 and by Gerencia Regional de Salud GRS963/A/2014 to M.L.R.G. We are particularly grateful to Mr. Leonides Alaiz for outstanding animal husbandry." The authors regret the errors.

Recombinant Lactobacillus strains have been constructed using gene splicing by overlap extension (SOE). Primers were designed of which one end of an amplified product contained complementary sequences for an end of other amplified fragment. For efficient matching, we used an asymmetric PCR step that was effective at generating an excess of strands that would anneal in the final PCR. CP12, a recombinant fragment consisting of the integrase gene and attachment site of the bacteriophage A2, was constructed and inserted into the genome of Lactobacillus casei ATCC 393, yielding Lb. casei ATCC 393::XCP12. Another recombinant Lb. casei strain was constructed, where the egfp gene was a part of the construction. The EGFP produced from Lb. casei ATCC 393::XCEGFP14 was detected by Western blot hybridization. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques for Lactobacillus species.

Background: Cyanobacteria are excellent model to understand the basic metabolic processes taking place in response to abiotic stress. The present study involves the characterization of a hypothetical protein Alr0765 of Anabaena PCC7120 comprising the CBS-CP12 domain and deciphering its role in abiotic stress tolerance.

Methods: Molecular cloning, heterologous expression and protein purification using affinity chromatography were performed to obtain native purified protein Alr0765. The energy sensing property of Alr0765 was inferred from its binding affinity with different ligand molecules as analyzed by FTIR and TNP-ATP binding assay. AAS and real time-PCR were applied to evaluate the iron acquisition property and cyclic voltammetry was employed to check the redox sensitivity of the target protein. Transcript levels under different abiotic stresses, as well as spot assay, CFU count, ROS level and cellular H2O2 level, were used to show the potential role of Alr0765 in abiotic stress tolerance. In-silico analysis of Alr0765 included molecular function probability analysis, multiple sequence analysis, protein domain and motif finding, secondary structure analysis, protein-ligand interaction, homologous modeling, model refinement and verification and molecular docking was performed with COFACTOR, PROMALS-3D, InterProScan, MEME, TheaDomEx, COACH, Swiss modeller, Modrefiner, PROCHECK, ERRAT, MolProbity, ProSA, TM-align, and Discovery studio, respectively.

Results: Transcript levels of alr0765 significantly increased by 20, 13, 15, 14.8, 12, 7, 6 and 2.5 fold when Anabaena PCC7120 treated with LC50 dose of heat, arsenic, cadmium, butachlor, salt, mannitol (drought), UV-B, and methyl viologen respectively, with respect to control (untreated). Heterologous expression resulted in 23KDa protein observed on the SDS-PAGE. Immunoblotting and MALDI-TOF-MS/MS, followed by MASCOT search analysis, confirmed the identity of the protein and ESI/MS revealed that the purified protein was a dimer. Binding possibility of Alr0765 with ATP was observed with an almost 6-fold increment in relative fluorescence during TNP-ATP binding assay with a λ max of 538 nm. FTIR spectra revealed modification in protein confirmation upon binding of Alr0765 with ATP, ADP, AMP and NADH. A 10-fold higher accumulation of iron was observed in digests of E. coli with recombinant vector after induction as compared to control, which affirms the iron acquisition property of the protein. Moreover, the generation of the redox potential of 146 mV by Alr0765 suggested its probable role in maintaining the redox status of the cell under environmental constraints. As per CFU count recombinant, E. coli BL21 cells showed about 14.7, 7.3, 6.9, 1.9, 3 and 4.9 fold higher number of colonies under heat, cadmium (CdCl2), arsenic (Na3AsO4), salt (NaCl), UV-B and drought (mannitol) respectively compared to pET21a harboring E. coli BL21 cells. Deterioration in the cellular ROS level and total cellular H2O2 concentration validated the stress tolerance ability of Alr0765. In-silico analysis unraveled novel findings and attested experimental findings in determining the role of Alr0765.

Conclusion: Alr0765 is a novel CBS-CP12 domain protein that maintains cellular energy level and iron homeostasis which provides tolerance against multiple abiotic stresses.

Keywords: Anabaena PCC7120; abiotic stress management; adenosyl ligand binding; hypothetical proteins; iron homeostasis; redox-active protein.

Adenylate kinases (ADK) are key enzymes that maintain the energetic balance in cellular compartments by catalyzing the reaction: AMP + ATP↔2 ADP. Here, we analyzed the chloroplast ADK 3 from the green alga, Chlamydomonas reinhardtii for the first time. This enzyme bears a C-terminal extension that is highly similar to the C-terminal end of the intrinsically disordered protein CP12 that plays a major role in the redox regulation of key enzymes of the Calvin-Benson cycle like glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase. The only other known example of a CP12-like extension is found in the GapB isoform of GAPDH, where it is responsible for the autonomous redox regulation of the higher plant A2 B2 GAPDH. In this study, we show that the CP12-like tail is not involved in the redox regulation of ADK 3, but contributes greatly to its stability, and is essential for the post-translational modification of the Cys221 residue by glutathione. This report highlights the fact that the C-terminal part of the CP12 protein can act as a moonlighting, intrinsically disordered module conferring additional capabilities to the proteins to which it is added.

Adenylate kinase (ADK, EC 2.7.4.3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13).

In our research to identify gene involved in the cuticle protein, we cloned a novel cuticle protein gene, ApCP13, from the Chinese oak silkmoth, Antheraea pernyi, larvae cDNA library. The ApCP13 gene encodes a 120 amino acid polypeptide with a predicted molecular mass of 13 kDa and a pI of 4.01, and is intron-less gene. The ApCP13 contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the ApCP13 cDNA is most homologous to another wild silkmoth, A. yamamai CP12 (86% protein sequence identity), followed by Bombyx mori LCP18 (35% protein sequence identity). Northern blot analysis revealed that the ApCP13 showed the epidermis-specific expression. This is the first report of cuticle protein gene in the wild silkmoth, A. pernyi.

BACKGROUND

Leaf and seed decoctions of Casimiroa spp. are used in Mexican traditional medicine to treat high blood pressure. Previous researches showed as Casimiroa extracts are able to induce relaxation of rat aortic and caudal arteries. To study the influence of the aging, we determined the vascular effect induced by extracts of Casimiroa edulis and Casimiroa pubescens in arterial tissues from young and old rats.

METHODS

The activity of Casimiroa edulis extracts: hexanic-leaf (Ce5), methanolic-leaf (Ce6), hexanic-seed (Ce7) and methanolic-seed (Ce8), and Casimiroa pubescens: hexanic-leaf (Cp9), methanolic-leaf (Cp10), hexanic-seed (Cp11) and methanolic-seed (Cp12) were investigated in precontracted caudal arteries of young (4 months) and old (20 months) rats.

RESULTS

The Casimiroa extracts tested at 20 μg/ml induced vasorelaxation in phenylephrine-precontracted arterial tissues both in young and old arterial tissues. Methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12) caused a higher relaxation in young than in old arterial tissues. Nifedipine (0.01 μM) did not change the vasorelaxation induced by Casimiroa edulis extract either in young and old rat arterial tissues.

CONCLUSIONS

The vasorelaxation induced by Casimiroa edulis and Casimiroa pubescens extracts is decreased from aging since the effects were higher in young than in old rat arterial tissues. However, the methanolic-seed extracts of both plant species induced a relevant vasorelaxation also in old arterial tissues. Thus the results support the traditional use of Casimiroa decoctions as antihypertensive, also in elderly.

Chitosan/nanohydroxyapatite composites based on scallop shells (CP12, CP14 and CP21) were prepared with different chitosan: nanohydroxyapatite ratios (1:2, 1:4 and 2:1, respectively). Nanohydroxyapatite (P), chitosan(C) and their composites were characterized by means of TGA, XRD, N2 adsorption/desorption analysis, SEM, Zeta potential and FTIR. The BET surface area ranged between 189 and 512 m2/g. Static adsorption of Hg+2 was tested for the effect of adsorbent dosage, pH, time and initial Hg+2 concentrations indicating that maximum static adsorption capacity was confirmed by CP12 (111.6 mg/g). Static adsorption well fitted with Langmuir adsorption isotherm and Pseudo-second order kinetic models. CP12 was selected for dynamic adsorption of Hg+2 considering the effect of bed height, flow rate and the effect of Hg+2 concentrations. Maximum dynamic adsorption capacity was confirmed at bed height of 3 cm, 2.0 mL/min flow rate and 300 mg/L as Hg+2 concentration with breakthrough time (tb) and exhaustion time (te) of 9 and 21 h. Yoon-Nelson and Thomas models best described the experimental Hg+2 breakthrough curve model. After static adsorption, EDTA solution confirmed the maximum desorption efficiency. The validity of CP12 was tested through three cycles of column dynamic adsorption-desorption.

Phosphoribulokinase (PRK) catalyses the ATP-dependent phosphorylation of ribulose 5-phosphate to give ribulose 1,5-bisphosphate. Regulation of this reaction in response to light controls carbon fixation during photosynthesis. Here, the crystal structure of PRK from the cyanobacterium Synechococcus sp. strain PCC 6301 is presented. The enzyme is dimeric and has an α/β-fold with an 18-stranded β-sheet at its core. Interestingly, a disulfide bond is found between Cys40 and the P-loop residue Cys18, revealing the structural basis for the redox inactivation of PRK activity. A second disulfide bond appears to rigidify the dimer interface and may thereby contribute to regulation by the adaptor protein CP12 and glyceraldehyde-3-phosphate dehydrogenase.

Isocyanic acid, HNCO, the imide of carbon dioxide, was prepared by reaction of stearic acid and potassium cyanate (KOCN) at 60 °C in a sealed, thoroughly dried reactor. Interestingly, its crystal structure, solved by X-ray single crystal diffraction at 123(2) K, shows a group-subgroup relation for the NCO- anion to carbon dioxide: (for CO2, cP12, Pa3̅, a = 5.624(2) Å, 150 K, C-O 1.151(2) Å; for HNCO, oP16, Pca21, a = 5.6176(9), b = 5.6236(8), c = 5.6231(7) Å, 123(2) K). Precise positions of H, N, C, and O were determined by DFT calculations with WIEN2k leading to interatomic distances C-O 1.17, C-N 1.22, N-H 1.03, and -N-H···N 2.14 Å, and the interatomic angle N-C-O 171°.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-known proverbial protein involved in various functions in vivo. The functional diversity of GAPDH from Dunaliella bardawil (DbGAPDH) may relate to the regulatory elements lying in the promoter at the transcriptional level. Using RT-PCR and RACE reactions, gapdh cDNA was isolated, and the full-length genomic sequence was obtained by LA-PCR-based genome walking. The full-length cDNA sequence was 1645 bp containing an 1128 bp putative open reading frame (ORF), which coded a 375 amino acids-deduced polypeptide whose molecular weight was 40.27 kDa computationally. Protein conserved domain search and structural computation found that DbGAPDH consists of two structural conserved domains highly homologous in most species; multiple sequence alignment discovered two positive charge residues (Lys164 and Arg 233), which play a critical role in the protein-protein interaction between GAPDH, phosphoribulokinase (PRK), and CP12. Phylogenetic analysis demonstrated that DbGAPDH has a closer relationship with analogues from algae and higher plants than with those from other species. In silico analysis of the promoter region revealed six potential regulatory elements might be involved in four hypothesized functions characterized by chloroplast GAPDH: oxygen-, light-, pathogen-, and cold-induced regulation. These results might supply some hints for the functional diversity mechanisms of DbGAPDH, and fresh information for further research to bridge the gap between our knowledge of DNA and protein structure and our understanding of functional biology in GAPDH regulation.

Fibronectin (FN) is a multimodular glycoprotein that is a critical component of the extracellular matrix (ECM) anlage during embryogenesis, morphogenesis, and wound repair. Our laboratory has previously described a family of FN-derived peptides collectively called "epiviosamines" that enhance platelet-derived growth factor-BB (PDGF-BB)-driven tissue cell survival, speed burn healing, and reduce scarring. In this study, we used an agarose drop outmigration assay to report that epiviosamines can enhance PDGF-BB-stimulated adult human dermal fibroblast (AHDF) outmigration in a dose-dependent manner. Furthermore, these peptides can, when delivered topically, stimulate granulation tissue formation in vivo. A thiol-derivatized hyaluronan hydrogel cross-linked with polyethyleneglycol diacrylate (PEGDA) was used to topically deliver a cyclized epiviosamine: cP12 and a cyclized engineered variant of cP12 termed cNP8 to porcine, full-thickness, excisional wounds. Both cP12 and cNP8 exhibited dose-dependent increases in granulation tissue formation at day 4, with 600 μM cNP8 significantly enhancing new granulation tissue compared to vehicle alone. In contrast to previous studies, this study suggests that epiviosamines can be used to increase granulation tissue formation without an exogenous supply of PDGF-BB or any cell-binding peptides. Thus, epiviosamine may be useful topically to increase granulation tissue formation in acute wounds.

The flagellated protozoan Trichomonas vaginalis (T. vaginalis) causes trichomoniasis, a reproductive tract infection, in humans. Trichomoniasis is the most common non-viral sexually transmitted disease worldwide. In addition to direct consequences such as infertility and abortion, there are indications that trichomoniasis favours development of prostate cancer and it has also been associated with increased risk of spreading human immunodeficiency virus and papillomavirus infections. Reports from around the world show that the rate of drug resistance in T. vaginalis is increasing, and therefore new therapeutic approaches have to be developed. Studying molecular biology of T. vaginalis will be quite helpful in identifying new drugable targets. RNAi is a powerful technique which allows biologist to specifically target gene products (i.e. mRNA) helping them in unravelling gene functions and biology of systems. However, due to lack of some parts of the required intrinsic RNAi machinery, the RNAi system is not functional in all orders of life. Here, by using synthetic siRNAs targeting two genes, i.e. α-actinin and cystein protease 12 (cp12), we demonstrate T. vaginalis cells are amenable to RNAi experiments conducted by extrinsic siRNAs. Electroporation of siRNAs targeting α-actinin or cp12 into T. vaginalis cells resulted in, respectively, 48-67% and 33-72% downregulation of the cognate transcripts compared to the T. vaginalis cells received siRNAs targeting GL2 luciferase as a control. This finding is helpful in that it demonstrates the potential of using extrinsically induced RNAi in studies on molecular biology of T. vaginalis such as those aiming at identifying new drug targets.

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.