Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and allowed us to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.     

Structural Basis for the ABO Blood-Group Dependence of Plasmodium falciparum Rosetting

The ABO blood group influences susceptibility to severe Plasmodium falciparum malaria. Recent evidence indicates that the protective effect of group O operates by virtue of reduced rosetting of infected red blood cells (iRBCs) with uninfected RBCs. Rosetting is mediated by a subgroup of PfEMP1 adhesins, with RBC binding being assigned to the N-terminal DBL1α₁ domain. Here, we identify the ABO blood group as the main receptor for VarO rosetting, with a marked preference for group A over group B, which in turn is preferred to group O RBCs. We show that recombinant NTS-DBL1α₁ and NTS-DBL1α₁-CIDR1γ reproduce the VarO-iRBC blood group preference and document direct binding to blood group trisaccharides by surface plasmon resonance. More detailed RBC subgroup analysis showed preferred binding to group A₁, weaker binding to groups A₂ and B, and least binding to groups A_x and O. The 2.8 Å resolution crystal structure of the PfEMP1-VarO Head region, NTS-DBL1α₁-CIDR1γ, reveals extensive contacts between the DBL1α₁ and CIDR1γ and shows that the NTS-DBL1α₁ hinge region is essential for RBC binding. Computer docking of the blood group trisaccharides and subsequent site-directed mutagenesis localized the RBC-binding site to the face opposite to the heparin-binding site of NTS-DBLα₁. RBC binding involves residues that are conserved between rosette-forming PfEMP1 adhesins, opening novel opportunities for intervention against severe malaria. By deciphering the structural basis of blood group preferences in rosetting, we provide a link between ABO blood grouppolymorphisms and rosette-forming adhesins, consistent with the selective role of falciparum malaria on human genetic makeup.     

Serum T-kininogen levels increase two to four months before death.

We have reported an accumulation of T-kininogen mRNA in the liver of aging Sprague-Dawley rats. T-kininogen is a cysteine proteinase inhibitor. Since a disruption of the intracellular protein degradation machinery is known to occur during senescence, we wished to further define the role of this protein in the aging process. As a first step, we have measured T-kininogen levels both in serum and within the liver. We have found that serum protein levels are indeed augmented during senescence, although not as dramatically as the mRNA (2.5-fold versus 8.3-fold). Immunocytochemistry, as well as Western blot analysis suggests that this is due to the presence of T-kininogen within hepatic cells in aged rats. Life-long dietary restriction, a known age-prolonging treatment, decreases the overexpression of the protein in 24-month-old rats. Later, diet-restricted animals still show an increased expression from the gene, the effect being delayed but not abolished by dietary manipulation. Interestingly, a longitudinal study indicated the existence of a positive correlation between the time of increase of serum T-kininogen and the time of death of the animal. Serum T-kininogen was found to increase 2.5-4 months before death.     

Structured fiber supports for gas phase biocatalysis

Pseudomonas cepaciae lipase adsorbed onto non-porous structured fiber supports in the form of woven fabrics, was used to catalyze hydrolysis and transesterification reactions in the gas phase. The enzyme adsorbed onto carbon fiber support exhibited much higher catalytic activity compared to the enzyme immobilized onto glass fiber carrier. The effect of temperature and relative humidity on reactions catalyzed by P. cepaciae lipase adsorbed onto structured fiber carbon support was studied in the gas system. Under the conditions investigated (up to 60 °C and 80% relative humidity), the immobilized enzyme showed a high thermostability and could be efficiently used to catalyze hydrolytic and transesterification reactions in continuous mode. Structured fiber supports, with a high specific surface area and a high mechanical resistance, showed a low-pressure drop during the passage of reactants through a reactor. The approach proposed in this study could be suitable for immobilization of a wide variety of enzymes.     

Intramolecular flip between two alternative forms of complex formed from a heme fragment and apoprotein of horse cytochrome c

The previous studies have shown that (a) noncovalent interactions of the ferro-heme fragment of residues 1-38 and apoprotein (1-104) of horse cytochrome c simultaneously and specifically form two isomeric complexes, types I and II resembling the native protein (the redundant residues flexibly protruding from the ordered structure); (b) the type II form but not type I appears to bind to CO; and (c) residues 39-55 are more flexible for type II form than type I (Parr, G. R., and Taniuchi, H. (1981) J. Biol. Chem. 256, 125-132). In the present study, we investigated 1) kinetics and thermodynamics of interconversion between type I and II forms of complex ferro-(1-38)-H.(1-104); 2) the properties of the CO binding population; 3) the rate of dissociation of complexes ferri- and ferro-(1-38)-H.(39-104) (mimicking type II form); and 4) thermal transition of the 695-nm absorption band and biological activity of complexes. The results indicate (a) interconversion between type I and II forms of complex ferro-(1-38)-H.(1-104) occurs without going through dissociation (t1/2 less than or equal to 12 min at 10 degrees C) and is associated with delta H (= -7.2 +/- 3.7 kcal/mol at 10 degrees C) favoring type I form and delta S (= 23 +/- 13 e.u. at 10 degrees C) favoring type II; (b) the CO-binding population correlates with type II; and (c) change from the ferrous to the ferric state of heme appears to perturb the thermodynamic relationship between type I and II forms. Interpreting the results and available evidence, we suggest that "intramolecular" flip between ferro-type I and ferro-type II forms would establish the Boltzmann distribution of these two distinctly different energy states, type I form having more strengthened interatomic interactions and type II more pronounced internal motion.