Mechanics of cellular adhesion to artificial artery templates

We are using polymer templates to grow artificial artery grafts in vivo for the replacement of diseased blood vessels. We have previously shown that adhesion of macrophages to the template starts the graft formation. We present a study of the mechanics of macrophage adhesion to these templates on a single cell and single bond level with optical tweezers. For whole cells, in vitro cell adhesion densities decreased significantly from polymer templates polyethylene to silicone to Tygon (167, 135, and 65 cells/mm(2)). These cell densities were correlated with the graft formation success rate (50%, 25%, and 0%). Single-bond rupture forces at a loading rate of 450 pN/s were quantified by adhesion of trapped 2-microm spheres to macrophages. Rupture force distributions were dominated by nonspecific adhesion (forces <40 pN). On polystyrene, preadsorption of fibronectin or presence of serum proteins in the cell medium significantly enhanced adhesion strength from a mean rupture force of 20 pN to 28 pN or 33 pN, respectively. The enhancement of adhesion by fibronectin and serum is additive (mean rupture force of 43 pN). The fraction of specific binding forces in the presence of serum was similar for polystyrene and polymethyl-methacrylate, but specific binding forces were not observed for silica. Again, we found correlation to in vivo experiments, where the density of adherent cells is higher on polystyrene than on silica templates, and can be further enhanced by fibronectin adsorption. These findings show that in vitro adhesion testing can be used for template optimization and to substitute for in-vivo experiments.     

D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

Role of phenylalanine B10 in plant nonsymbiotic hemoglobins

All plants contain an unusual class of hemoglobins that display bis-histidyl coordination yet are able to bind exogenous ligands such as oxygen. Structurally homologous hexacoordinate hemoglobins (hxHbs) are also found in animals (neuroglobin and cytoglobin) and some cyanobacteria, where they are thought to play a role in free radical scavenging or ligand sensing. The plant hxHbs can be distinguished from the others because they are only weakly hexcacoordinate in the ferrous state, yet no structural mechanism for regulating hexacoordination has been articulated to account for this behavior. Plant hxHbs contain a conserved Phe at position B10 (Phe(B10)), which is near the reversibly coordinated distal His(E7). We have investigated the effects of Phe(B10) mutation on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states. Kinetic and equilibrium constants for hexacoordination and ligand binding along with CO-FTIR spectroscopy, midpoint reduction potentials, and the crystal structures of two key mutant proteins (F40W and F40L) reveal that Phe(B10) is an important regulatory element in hexacoordination. We show that Phe at this position is the only amino acid that facilitates stable oxygen binding to the ferrous Hb and the only one that promotes ligand binding in the ferric oxidation states. This work presents a structural mechanism for regulating reversible intramolecular coordination in plant hxHbs.     

Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] < [Ca(2+)], [Mg(2+)], [Mn(2+)] < [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.     

An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.     

Body cooling between two bouts of exercise in the heat enhances subsequent performance

The purpose was to assess whether body cooling between 2 bouts of exercise in the heat enhances performance during the second exercise session. Using a random, crossover design, 15 subjects (3 women, 12 men; 28 +/- 2 years, 180 +/- 2 cm, 69 +/- 2.3 kg) participated in all 3 trials. Subjects ran 90 minutes on hilly trails in a hot environment (approximately 27 degrees C) before 12 minutes of either cold water immersion (CWI; 13.98 degrees C), ice water immersion (IWI; 5.23 degrees C), or a mock treatment (MT) of sitting in a tub with no water (29.50 degrees C). After immersion, subjects ran a 2-mile race. CWI had faster (p < 0.05) performance time (725 seconds) than MT (769 seconds). CWI and IWI had significantly (p < 0.05) lower rectal temperatures postimmersion than MT as well as postrace (p < 0.05). Heart rate also remained significantly lower (p < 0.05) during the CWI and IWI trials for the first half of the race. In conclusion, CWI enhances performance (6% improvement in race time) in the second bout of exercise, supporting its potential role as an ergogenic aid in athletic performance.     

The contribution of DNA repair and antioxidants in determining cell type-specific resistance to oxidative stress

The aims of this study were; (i) to elucidate the mechanisms involved in determining cell type-specific responses to oxidative stress and (ii) to test the hypothesis that cell types which are subjected to high oxidative burdens in vivo, have greater oxidative stress resistance. Cultures of the retinal pigment epithelium (RPE), corneal fibroblasts, alveolar type II epithelium and skin epidermal cells were studied. Cellular sensitivity to H2O2 was determined by the MTT assay. Cellular antioxidant status (CuZnSOD, MnSOD, GPX, CAT) was analyzed with enzymatic assays and the susceptibility and repair capacities of nuclear and mitochondrial genomes were assessed by QPCR. Cell type-specific responses to H2O2 were observed. The RPE had the greatest resistance to oxidative stress (P>0.05; compared to all other cell types) followed by the corneal fibroblasts (P < 0.05; compared to skin and lung cells). The oxidative tolerance of the RPE coincided with greater CuZnSOD, GPX and CAT enzymatic activity (P < 0.05; compared to other cells). The RPE and corneal fibroblasts both had up-regulated nDNA repair post-treatment (P < 0.05; compared to all other cells). In summary, variations in the synergistic interplay between enzymatic antioxidants and nDNA repair have important roles in influencing cell type-specific vulnerability to oxidative stress. Furthermore, cells located in highly oxidizing microenvironments appear to have more efficient oxidative defence and repair mechanisms.     

Convergence of vitamin D and retinoic acid signalling at a common hormone response element

Although 1,25-dihydroxyvitamin D3 (1,25D3) and retinoic acid (RA) have distinct developmental and physiological roles, both regulate the cell cycle. We provide molecular and genomic evidence that their cognate nuclear receptors regulate common genes through everted repeat TGA(C/T)TPyN8PuG(G/T)TCA (ER8) response elements. ER8 motifs were found in the promoters of several target genes of 1,25D3 and/or RA. Notably, an element was characterized in the cyclin-dependent kinase (CDK) inhibitor p19ink4d gene, and 1,25D3- or RA-induced p19INK4D) expression. P19ink4d knockdown together with depletion of p27kip1, another CDK inhibitor regulated by 1,25D3 and RA, rendered cells resistant to ligand-induced growth arrest. Remarkably, p19INK4D-deficient cells showed increased autophagic cell death, which was markedly enhanced by 1,25D3, but not RA, and attenuated by loss of p27KIP1. These results show a limited crosstalk between 1,25D3 and RA signalling by means of overlapping nuclear receptor DNA binding specificities, and uncover a role for p19INK4D in control of cell survival.