Molecular Dynamics Studies of Proton Transport in Hydrogenase and Hydrogenase Mimics.

There is extensive interest in hydrogenases based on their ability to rapidly and efficiently interconvert H2 with protons and electrons, and their (typically) superior function relative to molecular mimics. Understanding the function of enzymes is one approach to implementing design features to make better catalysts and is an approach we have implemented in our work. In this review, we will discuss our efforts to develop design principles from enzymes, with specific focus on proton transport. We will also present computational studies of the mimics we have investigated with similar methodologies. We will discuss the mechanisms used by small scaffolds on molecular mimics which in many cases are surprisingly similar to those used by nature, while in other cases, computational analysis allowed us to reveal an unexpected role. Computational methods provide one of the best ways, and in some cases, the only way, to gain insight into the mechanistic details of enzymes. In this review, we illustrate the general computational strategy we used to study the proton pathway of [FeFe]-hydrogenase, and the similar strategy to investigate small molecules. We present the main results we obtained and how our computational work stimulated or worked in concert with experimental investigations. We also focus on estimation of errors and convergence of properties in the simulations. These studies demonstrate the powerful results that can be obtained by the close pairing of experimental and theoretical approaches.

Elevated levels of soluble E-selectin in diabetic patients with severe symptomatic peripheral arterial occlusive disease requiring angioplasty. A possible role in diabetic vascular disease?

BACKGROUND: To study levels of E-selectin in patients with peripheral arterial occlusive disease who were undergoing percutaneous transluminal angioplasty. METHODS: Experimental design and setting: Cross sectional case control study, conducted in a teaching hospital. SUBJECTS: Seven patients with type-2 (non-insulin dependent) diabetes mellitus undergoing transluminal angioplasty for symptomatic peripheral arterial occlusive disease, had blood tests for sE-selectin measurement, and were compared to a similar groups of age and sex matched non-diabetic patients with arteriopathy who are undergoing the same procedure. Also evaluated were a group of diabetic patients and healthy non-diabetics with no peripheral arterial disease. RESULTS: The levels of sE-selectin in the two diabetic groups were significantly higher than the non-diabetic groups measuring at 77 ng/ml (53-120) and 79 ng/ml (43-98) (median, range) vs 54 ng/ml (24-104) in the non-diabetic with arteriopathy, and 42 ng/ml (35-66) in the normal healthy controls, p<0.04, p<0.003 respectively, Mann Whitney "U" test. CONCLUSIONS: We have demonstrated significantly high values of soluble E-selectin in patients with diabetes mellitus requiring angioplasty for symptomatic peripheral arterial occlusive disease. This suggests that sE-selectin may be involved in the diabetic angiopathic process. It may act as a precursor for smooth muscle proliferation.

Calves chronically implanted with a total artificial heart as a pharmacological model.

Pharmacological therapy for congestive heart failure includes drugs that have both inotropic and vasoactive effects, although it is sometimes difficult to differentiate between the two effects. An animal with an implanted total artificial heart (TAH) allows the investigation of the vascular effect of these drugs in the absence of the effect on the myocardium. An advantage of the TAH model is its sensitivity to changes in right and left ventricular preload and afterload. Four instrumented TAH calves were given vasoactive drugs and the response was compared to control. Epinephrine, dopamine, isoproterenol, and nitroprusside were selected because of the predictability of their responses. Epinephrine caused a significant increase in systemic vascular resistance (SVR), and dopamine caused a significant increase in Pulmonary vascular resistance (PVR) and Isoproterenol caused a significant decrease in PVR. TAH implanted calves can thus serve as a pharmacological model to study the vascular response, which may be useful in investigation of new agents with inotropic and vascular effects.

Mineral association changes the secondary structure and dynamics of murine amelogenin.

Amelogenin is one of the key protein constituents responsible for the exquisite organization of the calcium phosphate crystals in enamel. Amelogenin forms into nanospheres in solution, while its association with hydroxyapatite is also essential to enamel development. Structural information of full-length amelogenin in either of these physiologically important forms has the potential to provide mechanistic information; however, these data are limited because of the difficulty of determining the structure of large protein complexes and proteins bound to surfaces. To obtain structural insights into amelogenin during these early stages of enamel development, we used a lysine-specific (13)C-, (15)N-labeled sample of murine amelogenin to provide insight into the structure of the hydroxyapatite (HAP)-binding domains of the protein. A combination of one-and two-dimensional solid-state NMR experiments was used to obtain molecular-level insights into the secondary structure and dynamics of full-length amelogenin within a nanosphere-gel and on the surface of HAP. Regions of amelogenin that appear to be primarily random coil in the nanosphere-gel adopt a ß-strand structure and are less mobile with HAP binding, indicative of a structural switch upon binding that may be important in the role of amelogenin in enamel development.

Channelized ice melting in the ocean boundary layer beneath Pine Island Glacier, Antarctica.

Ice shelves play a key role in the mass balance of the Antarctic ice sheets by buttressing their seaward-flowing outlet glaciers; however, they are exposed to the underlying ocean and may weaken if ocean thermal forcing increases. An expedition to the ice shelf of the remote Pine Island Glacier, a major outlet of the West Antarctic Ice Sheet that has rapidly thinned and accelerated in recent decades, has been completed. Observations from geophysical surveys and long-term oceanographic instruments deployed down bore holes into the ocean cavity reveal a buoyancy-driven boundary layer within a basal channel that melts the channel apex by 0.06 meter per day, with near-zero melt rates along the flanks of the channel. A complex pattern of such channels is visible throughout the Pine Island Glacier shelf.

Structure and dynamics of hydrated statherin on hydroxyapatite as determined by solid-state NMR.

Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composite technologies. Here, we have used solid-state NMR techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Backbone secondary structure for the N-terminal dodecyl region was determined using a combination of homonuclear and heteronuclear dipolar recoupling techniques. Both sets of experiments indicate the N-terminus is alpha-helical in character with the residues directly binding to the HAP being stabilized in the alpha-helical conformation by the presence of water. Dynamic NMR studies demonstrate that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of this domain are mobile and thus weakly interacting with the mineral surface. The direct binding footprint of statherin is thus localized to the negatively charged N-terminal pentapeptide sequence. Study of a site-directed mutant demonstrated that alteration of the only anionic side chain outside of this domain did not affect the dynamics of statherin on the HAP surface, suggesting that it does not play an important role in HAP binding.

Insertion of CO2, CS2, and COS into iron(II)-hydride bonds.

The reactions between cis-Fe(dmpe)2H2 (dmpe = Me2PCH2CH2PMe2) (1) or cis-Fe(PP3)H2 (PP3 = P(CH2CH2PMe2)3) (2) and carbon dioxide (CO2), carbon disulfide (CS2), and carbonyl sulfide (COS) are investigated. At 300 K, additions of CO2 (1 atm), CS2 (2 equiv), and COS (1 atm) to 1 result in the formation of a stable transformato hydride, trans-Fe(dmpe)2(OCHO)H (3a), a trans-dithioformato hydride, trans-Fe(dmpe)2(SCHS)H (4a), and a trans-thioformato hydride, trans-Fe(dmpe)2(SCHO)H (5a), respectively. When CS2 and COS are added to cis-Fe(dmpe)2H2 at 195 K, a cis-dithioformato hydride, 4b, and a cis-thioformato hydride, 5b, respectively, are observed as the initially formed products, but there is no evidence of the corresponding cis-formato hydride upon addition of CO2 to cis-Fe(dmpe)2H2. Additions of excess CO2, CS2, and COS to 1 at lower temperatures (195-240 K) result in the formation of a trans-bis(formate), trans-Fe(dmpe)2(OCHO)2 (3b), a trans-bis(dithioformate), trans-Fe(dmpe)2(SCHS)2 (4c), and a cis-bis(thioformate), cis-Fe(dmpe)2(SCHO)2 (5c), respectively. trans-Fe(dmpe)2(SCHO)2 (5d) is prepared by the addition of excess COS at 300 K. Additions of CO2 (1 atm), CS2 (0.75 equiv), and COS (1 atm) to 2 at 300 K result in the formation of a thermally stable, geometrically constrained cis-formato hydride, cis-Fe(PP3)(OCHO)H (6a), a cis-dithioformato hydride, cis-Fe(PP3)(SCHS)H (7a), and a cis-thioformato hydride, cis-Fe(PP3)(SCHO)H (8a), respectively. Additions of excess CO2 and COS to 2 yield a cis-bis(formate), cis-Fe(PP3)(OCHO)2 (6b), and a thermally stable cis-bis(thioformate), cis-Fe(PP3)(SCHO)2 (8b), respectively. All complexes are characterized by multinuclear NMR spectroscopy, with IR spectroscopy and elemental analyses confirming structures of thermally stable complexes where possible. Complexes 3b and 5a are also characterized by X-ray crystallography.

Factors influencing the accuracy of the cardiac output monitoring and diagnostic unit for pneumatic artificial hearts.

The Cardiac Output Monitor and Diagnostic Unit (COMDU) has been the most widely used method to noninvasively determine cardiac output in pneumatic ventricles for the past 10 years. Clinical observation has suggested a discrepancy between the COMDU and expected cardiac outputs. In vivo tests verified and quantified this error. The error sources were examined using in vitro test conditions, with both the inflow and outflow, as well as COMDU flow readings, being analyzed. Transducer and calibration error sources were also identified, and the accuracy of the method for determining cardiac output for the in vitro test conditions was quantified. With a more accurate calibration scheme, the in vitro average error was reduced from -16.2% (range of 0.1% to -41.1%) to 0.1% (range 4.8% to -3.65). The major error sources were identified as missed inflow, transducer calibration and drift, and system variance.

Radical and non-radical mechanisms for alkane oxidations by hydrogen peroxide-trifluoroacetic acid.

The oxidation of cyclohexane by the H2O2-trifluoroacetic acid system is revisited. Consistent with a previous report (Deno, N.; Messer, L. A. Chem. Comm. 1976, 1051), cyclohexanol forms initially but then esterifies to cyclohexyl trifluoroacetate. Small amounts of trans-1,2-cyclohexadiyl bis-(trifluoroacetate) also form. Although these products form irrespective of the presence or absence of O2, dual mechanisms are shown to operate. In the absence of O2, the dominant mechanism is a radical chain reaction that is propagated by CF3. abstracting H from C6H12 and SH2 displacement of C6H11. on CF3CO2OH. The intermediacy of C6H11. and CF3. is inferred from production of CHF3 and CO2 along with cyclohexyl trifluoroacetate, or CDF3 when cyclohexane-d12 is used. In the presence of O2, fluoroform and CO2 are suppressed, the reaction rate slows, and the rate law approaches second order (first order in peracid and in C6H12). Trapping of cyclohexyl radicals by quinoxaline is inefficient except at elevated (approximately 75 degrees C) temperatures. Fluoroform and CO2, telltale evidence for the chain pathway, were not produced when quinoxaline was present in room temperature reactions. These observations suggest that a parallel, nonfree radical, oxenoid insertion mechanism dominates when O2 is present. A pathway is discussed in which a biradicaloid-zwiterionic transition state is attained by hydrogen transfer from alkane to peroxide oxygen with synchronous O-O bond scission.

Chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion.

Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.

Particulate Air Pollution in Mexico City: A Collaborative Research Project.

PM10, PM25, precursor gas, and upper-air meteorological measurements were taken in Mexico City, Mexico, from February 23 to March 22, 1997, to understand concentrations and chemical compositions of the city's particulate matter (PM). Average 24-hr PM10 concentrations over the period of study at the core sites in the city were 75 H g/m3. The 24-hr standard of 150 µ g/m3 was exceeded for seven samples taken during the study period; the maximum 24-hr concentration measured was 542 µ g/m3. Nearly half of the PM10 was composed of fugitive dust from roadways, construction, and bare land. About 50% of the PM10 consisted of PM2.5, with higher percentages during the morning hours. Organic and black carbon constituted up to half of the PM2.5. PM concentrations were highest during the early morning and after sunset, when the mixed layers were shallow. Meteorological measurements taken during the field campaign show that on most days air was transported out of the Mexico City basin during the afternoon with little day-to-day carryover.