Bioinspired ligand designs for cobalt, iron and manganese complexes : understanding mono-iron hydrogenase (Hmd)

Mono-iron hydrogenase is one of three types of hydrogenases, catalyzing reversible hydride transfer to the substrate (methenyl-H₄MPT⁺) by heterolytically cleaving molecular hydrogen into a proton and a hydride. The key features of the enzyme’s active site include a pyridone moiety, an acyl unit and facial ligation of C [superscript acyl] N [superscript pyridone] S [superscript Cys] donors. Each feature was independently incorporated into a selected ligand system (Schiff-base N4, pincer and thianthrene scaffold, respectively), in efforts to i) develop possible bioinspired catalysts for H₂ activation using earth abundant metals (iron, cobalt, manganese) and ii) make synthetic models of the enzyme active site for deeper understanding of the architecture of the active site and catalytic mechanism. Synthetic routes for making pyridone-based Schiff-base N4 and NNS type ligands were explored: although not isolated, the ligands were spectroscopically detected. On the other hand, the simpler version—pyridine-based Schiff-base N4 ligands—afforded dinuclear cobalt complexes upon metalations with cobalt(II) precursors. Depending on the length of the diamine-linker as well as the substituents on the pyridine rings, either spontaneous O₂ activation or B–F activation was observed, yielding μ-peroxo dicobalt(III) complexes or μ-fluoride bridged dicobalt(II) complexes, respectively. In particular, two μ-fluoride bridged dicobalt complexes showed antiferromagnetic coupling between the two cobalt(II) centers. From the pincer ligands featuring the unique acyl moiety within C [superscript acyl] N [superscript pyridine] S [superscript thioehter] and C [superscript acyl] N [superscript pyridine] P [superscript Ph2] donor set, the (expected) meridional and an (unexpected) facial iron-acyl complexes were isolated, respectively. Upon deprotonation (pyridine→pyridinate de-aromatization), both complexes showed reactivity towards H₂ activation; no evidence for hydride-transfer was observed. For the facial ligation, thianthrene-scaffolded manganese system was examined as a more flexible version of the anthracene-scaffolded systems. Preliminary results of the kinetic studies support the correlation between the flexibility of the scaffold and the reactivity of the metal complex, without greatly altering the electronic environment of the metal center.Chemistr

Risk Aversion or Risk Management?: How Measures of Risk Aversion Affect Firm Entry and Firm Survival

The link between measured risk aversion and the decision to become an entrepreneur is well established, but the link between risk preferences and entrepreneurial success is not. Standard theoretical models of occupational choice under uncertainty imply a positive correlation between an individual’s degree of risk aversion and the expected return from an entrepreneurial venture at the time of entry. Because the expected return is the risk neutral equivalent value, a higher expected return implies a higher survival probability, and so more risk averse entrepreneurs should survive more frequently than their less risk averse counterparts. We test that prediction using successive entry cohorts of young entrepreneurs in the National Longitudinal Survey of Youth 1979 (NLSY79). The empirical results soundly reject the prediction: the most successful entrepreneurs are the least risk averse. This surprising finding calls into question the interpretation of common measures of risk aversion as measures of taste for risk. Instead, measured risk attitudes perform as if they are indicators of entrepreneurial ability– the least risk averse are apparently those who can best assess and manage risks. Indeed, our interpretation is consistent with the work of recent experimental studies that find that the less risk averse have higher cognitive ability.

Recovery of endurance running capacity: effect of carbohydrate-protein mixtures

Including protein in a carbohydrate solution may accelerate both the rate of glycogen storage and the restoration of exercise capacity following prolonged activity. Two studies were undertaken with nine active men in study A and seven in study B. All participants performed 2 trials, each involving a 90 min run at 70% VO2max followed by a 4 h recovery. During recovery, either a 9.3% carbohydrate solution (CHO) or the same solution plus 1.5% protein (CHO-PRO) was ingested every 30 min in volumes providing either 1.2 g CHO · kg-1 · h-1 (study A) or 0.8 g CHO · kg-1 · h-1 (study B). Exercise capacity was then assessed by run time to exhaustion at 85% VO2max. Ingestion of CHO-PRO elicited greater insulinemic responses than CHO (P less than or equal to 0.05) but with no differences in run times to exhaustion. Within the context of this experimental design, CHO and CHO-PRO restored running capacity with equal effect

Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo.

Members of the TGF-beta superfamily appear to modulate mesenchymal differentiation, including the processes of cartilage and bone formation. Nothing is yet known about the function of the TGF-beta-related factor vgr-1, also called bone morphogenetic protein-6 (BMP-6), and only limited studies have been conducted on the most closely related factors BMP-5, osteogenic protein-1 (OP-1) or BMP-7, and OP-2. Because vgr-1 mRNA has been localized in hypertrophic cartilage, this factor may play a vital role in endochondral bone formation. We developed antibodies to vgr-1, and documented that vgr-1 protein was expressed in hypertrophic cartilage of mice. To further characterize the role of this protein in bone differentiation, we generated CHO cells that overexpressed recombinant murine vgr-1 protein. Western blot analysis documented that recombinant vgr-1 protein was secreted into the media and was proteolytically processed to yield the mature vgr-1 molecule. To assess the biological activity of recombinant vgr-1 in vivo, we introduced the vgr-1-expressing CHO cells directly into the subcutaneous tissue of athymic nude mice. CHO-vgr-1 cells produced localized tumors, and the continuous secretion of vgr-1 resulted in tumors with a strikingly different gross and histological appearance as compared to the parental CHO cells. The tumors of control CHO cells were hemorrhagic, necrotic, and friable, whereas the CHO-vgr-1 tumors were dense, firm, and fibrotic. In contrast with control CHO tumors, the nests of CHO-vgr-1 tumor cells were surrounded by extensive connective tissue, which contained large regions of cartilage and bone. Further analysis indicated that secretion of vgr-1 from the transfected CHO tumor cells induced the surrounding host mesenchymal cells to develop along the endochondral bone pathway. These findings suggest that endochondral bone formation

Carbohydrate gel ingestion significantly improves the intermittent endurance capacity, but not sprint performance, of adolescent team games players during a simulated team games protocol

The aim of this study was to investigate the influence of ingesting a carbohydrate (CHO) gel on the intermittent endurance capacity and sprint performance of adolescent team games players. Eleven participants [mean age 13.5 ± 0.7 years, height 1.72 ± 0.08 m, body mass (BM) 62.1 ± 9.4 kg] performed two trials separated by 3–7 days. In each trial, they completed four 15 min periods of part A of the Loughborough Intermittent Shuttle Test (LIST), followed by an intermittent run to exhaustion (part B). In the 5 min pre-exercise, participants consumed 0.818 mL kg−1 BM of a CHO or a non-CHO placebo gel, and a further 0.327 mL kg−1 BM every 15 min during part A of the LIST (38.0 ± 5.5 g CHO h−1 in the CHO trial). Intermittent endurance capacity was increased by 21.1% during part B when the CHO gel was ingested (4.6 ± 2.0 vs. 3.8 ± 2.4 min, P < 0.05, r = 0.67), with distance covered in part B significantly greater in the CHO trial (787 ± 319 vs. 669 ± 424 m, P < 0.05, r = 0.57). Gel ingestion did not significantly influence mean 15 m sprint time (P = 0.34), peak sprint time (P = 0.81), or heart rate (P = 0.66). Ingestion of a CHO gel significantly increases the intermittent endurance capacity of adolescent team games players during a simulated team games protocol

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation

Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation

The polarized expression of Na+,K+-ATPase in epithelia depends on the association between beta-subunits located in neighboring cells

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta(1)-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta(1)-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta(1)-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta(1)-subunit forces CHO-beta cells to coexpress endogenous a-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog P,-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells

Carbohydrate Mouth Rinse Improves 1.5 h Run Performance: Is There a Dose-Effect?

There is a substantial body of recent evidence showing ergogenic effects of carbohydrate (CHO) mouth rinsing on endurance performance. However, there is a lack of research on the dose-effect and the aim of this study was to investigate the effect of two different concentrations (6% and 12% weight/volume, w/v) on 90 minute treadmill running performance. Seven active males took part in one familiarization trial and three experimental trials (90-minute self-paced performance trials). Solutions (placebo, 6% or 12% CHO-electrolyte solution, CHO-E) were rinsed in the mouth at the beginning, and at 15, 30 and 45 minutes during the run. The total distance covered was greater during the CHO-E trials (6%, 14.6 ± 1.7 km; 12%, 14.9 ± 1.6 km) compared to the placebo trial (13.9 ± 1.7 km, P 0.05). There were no between trial differences (P > 0.05) in ratings of perceived exertion (RPE) and feeling or arousal ratings suggesting that the same subjective ratings were associated with higher speeds in the CHO-E trials. Enhanced performance in the CHO-E trials was due to higher speeds in the last 30 minutes even though rinses were not provided during the final 45 minutes, suggesting the effects persist for at least 20-45 minutes after rinsing. In conclusion, mouth rinsing with a CHO-E solution enhanced endurance running performance but there does not appear to be a dose-response effect with the higher concentration (12%) compared to a standard 6% solution

BRST symmetry of SU(2) Yang-Mills theory in Cho--Faddeev--Niemi decomposition

We determine the nilpotent BRST and anti-BRST transformations for the Cho--Faddeev-Niemi variables for the SU(2) Yang-Mills theory based on the new interpretation given in the previous paper of the Cho--Faddeev-Niemi decomposition. This gives a firm ground for performing the BRST quantization of the Yang--Mills theory written in terms of the Cho--Faddeev-Niemi variables. We propose also a modified version of the new Maximal Abelian gauge which could play an important role in the reduction to the original Yang-Mills theory.Comment: 11 pages, no figure; Introduction improved, 3 references adde