Characterization of dentine structure in three dimensions using FIB-SEM.

All biological tissues are three dimensional and contain structures that span a range of length scales from nanometres through to hundreds of millimetres. These are not ideally suited to current three-dimensional characterization techniques such as X-ray or transmission electron tomography. Such detailed morphological analysis is critical to understanding the structural features relevant to tissue function and designing therapeutic strategies intended to address structural deficiencies encountered in pathological states. We show that use of focused ion beam milling combined with scanning electron microscopy can provide three-dimensional information at nanometre resolution from biologically relevant volumes of material, in this case dentine.

Redox concerns in the use of acellular hemoglobin-based therapeutic oxygen carriers: the role of plasma components.

Within the past decade, most research efforts in the red blood cell substitute area have revolved about the development of acellular hemoglobin-based oxygen carriers (HBOC) as clinical replacements and/or augmentation of human blood's carrying and delivery function. A major requirement for all HBOC is the maintenance of the heme-Fe+2 in this reduced state for normal physiological behavior. Oxidation of hemoglobin results in the formation of methemoglobin (heme-Fe+3). MetHb is unable to bind oxygen thus effectively lowering the carrying capacity of the Hb-based substitute. In addition, met Hb gives rise to free radicals that have the potential to cause endothelial and surrounding tissue damage. Results of this study suggest that the normal endogenous reducing agents of human plasma have the capacity to provide redox protection and stability to specific acellular-types of HBOC. The effectiveness of these reducing agents may be related to the formal reduction potential of the HBOC being considered. The choice of buffer for HBOC storage is critical and specific to the HBOC product.

Alteration of redox stability of hemoglobins A and S by biological buffers.

Several organic and inorganic buffers, including Hepes, Tris and potassium phosphate, are often used for the maintenance of pH in hemoglobin solution work. It has often been assumed these buffers do not significantly affect the solution stability of these proteins. This investigation has focused on the effect these buffers have on the redox stability of the heme-iron, [Fe+2-->Fe+3], of hemoglobins A and sickle cell S (beta 6 glu-->val) over extended periods of time. Initial results indicate that: 1) the use of different buffers under similar ionic strength conditions at pH 7.0 affect conformational changes that produce substantial differences in redox stability between Hb A and Hb S; 2) spectral analysis of these hemoglobins in the visible region (700-500 nm) at 37 degrees C for extended periods of time indicated that a slower rate of autoxidation is observed with a Hepes buffered system over a wide ionic strength range than for either Tris or potassium phosphate buffers; 3) Hb S autoxidized at a faster rate in the presence of each of these buffers than Hb A; and 4) in contrast to Hb A, Hb S showed extensive hemichrome formation after 15 hr in the presence of potassium phosphate buffer.

A first evaluation of the natural high molecular weight polymeric Lumbricus terrestris hemoglobin as an oxygen carrier.

Lumbricus terrestris hemoglobin (LtHb), an unusually stable Hb (MW approximately 4x10(6) Da) with respect to dissociation and oxidation, circulates extracellularly in the earthworm and at neutral pH exhibits oxygen affinity and cooperativity similar to that of human HbA. Results suggest that LtHb may serve as a model for a high molecular weight extracellular oxygen carrier. Mice and a rat model partially exchanged with LtHb showed no apparent behavioral and physical changes. 31P NMR spectroscopy of perfused guinea pig hearts, used to assess phosphocreatine levels as an indication of the ability of LtHb to serve as an oxygen carrier to the heart, demonstrated that LtHb provides oxygen to the tissue and maintains the energy metabolism significantly better than the control non-Hb perfusion media. One day after infusion, video enhanced microscopy imaging of the mice cremaster muscle vasculature reveals temporal adhesion of leukocytes to the endothelial walls with temporal infiltration of leukocytes to the surrounding tissue, correlated with dosage. Exchanged mice rechallenged with LtHb show no overt allergic response or death. Further evaluation of this natural extracellular Hb as a potential polymeric Hb blood substitute/perfusion agent is warranted.

Applying a relative ranking model to the self-assessment of extended performances.

BACKGROUND: Accurate self-assessment is an important but underdeveloped skill in medicine that, in the past, has received little formal attention from educators. METHOD: Following an orthopedic rotation, twenty-five orthopedic surgery residents performed a self-assessment task for ten skills using a new relative ranking method, in which an individual's skills are ranked relative to each other rather than being compared to the individual's peers. Supervising faculty assessed residents using the same instrument. Faculty inter-rater reliability was measured and comparisons were made between each resident's self-assessment and the faculty assessments using Spearman rank order correlation coefficients. RESULTS: The mean correlation between faculty rating the same resident was 0.27 (sd = 0.49). The mean correlation between resident and faculty rankings was 0.20 (sd = 0.38), but was higher for junior residents (0.33) than for senior residents (0.12), apparently because senior residents do not alter their self-assessments while faculty change their assessments of senior residents. CONCLUSIONS: Consistent with the literature in other fields, we find that self-assessment is poor among surgical trainees when they are asked to assess their own performance over an extended time period.

Analysis of shorthorn sculpin antifreeze protein stereospecific binding to (2-1 0) faces of ice.

In this paper we report the results of our studies on the stereospecific binding of shorthorn sculpin antifreeze protein (AFP) to (2 -1 0) secondary prism faces of ice. Using ice crystal growth and etching techniques together with molecular modeling, molecular dynamics, and energy minimization, we explain the nature of preferential binding of shorthorn sculpin AFP along the [1 2 2] direction on (2- 1 0) planes. In agreement with ice etching studies, the mechanism of preferential binding suggested by molecular modeling explains why the binding of shorthorn sculpin AFP occurs along [1 2 2] and not along its mirror symmetry-related direction [-1 -2 2] on (2 -1 0). This binding mechanism is based on the protein-crystal surface enantioselective recognition that utilizes both alpha-helical protein backbone matching to the (2 -1 0) surface topography and matching of side chains of polar/charged residues with specific water molecule positions in the ice surface. The mechanisms of winter flounder and shorthorn sculpin antifreeze binding to ice are compared.

Spectral analysis of Fe(III)-complex reduction by hemoglobin: possible mechanisms of interaction.

1. Hemoglobin is capable of electron transfer to Fe(III)-complexes of ATP, EDTA, NTA, and citrate leading to formation of reduced Fe(II) and its concurrent release from these chelating compounds as evident in the formation of a Fe(II)-Tris 2,2' bipyridine complex. 2. Multi-component analysis of kinetic spectra in the visible region (700-500 nm) has permitted a determination of the effect of various chelating molecules bound to Fe(III), pH, the effects of ionic strength, temperature, and the molecular nature of the Fe(III)-complex on reaction rates. 3. We have examined and compared the reactivities of normal adult hemoglobin A (alpha 2 beta 2) to reduce these Fe(III)-complexes and suggest possible mechanism(s) for the electron transfer process.

Stability of a potential blood substitute, HbXL99 alpha, under high pressure.

One important criteria for a plasma circulating hemoglobin blood substitute is resistance to subunit dissociation. For this reason, cross-linked hemoglobins (with low oxygen affinities) are being specifically designed to serve as potential blood substitutes. An example is HbXL99 alpha, cross-linked between the alpha-subunits [PNAS (1987) 84:7280]. In the study presented here, the effects of up to 2 kilobars of pressure on the intrinsic fluorescence of HbXL99 alpha, HbA, and myoglobin were compared. Hemoglobin solutions were studied between 0.01-0.1g% in potassium phosphate or Hepes buffers, pH 7.4. Results show HbA exhibits a decrease in fluorescence intensity as a function of pressure. In contrast, HbXL99 alpha as well myoglobin (a monomer) show essentially no significant intrinsic fluorescence changes as a function of pressure. These results suggest that HbXL99 alpha is stable as a tetramer up to approximately 2 kilobars of pressure. In addition, high pressure intrinsic fluorescence studies provide a suitable technique for determining the subunit stability of hemoglobins.

Alteration of tryptophan fluorescence properties upon dissociation of Lumbricus terrestris hemoglobin.

Fluorescence analysis has been used to study dissociation of the dodecameric 3.8 kDa Lumbricus terrestris hemoglobin. Since tryptophan intrinsic fluorescence has been used as a reporter group to study Lumbricus hemoglobin, it is of interest to study dissociation perturbed properties of the tryptophan residues. Shifts in the fluorescence emission maximum to longer wavelengths upon dissociation at pH 9.2 suggest that tryptophans buried at the subunit interface(s) become more exposed. Fluorescence lifetime and quenching studies are employed in this present investigation as a means to confirm the location of tryptophan residues at the subunit interfaces. Acrylamide titration (to 2.5 M) indicate only a fraction of the residues can be quenched at either pH. At pH 7.0, the Stern-Volmer plot has downward curvature, while at pH 9.2 there is slight upward curvature, again indicating a change in environment. The intrinsic fluorescence decay requires at least four exponentials at both pHs. The mean fluorescence lifetime of CO Lumbricus hemoglobin increases from 1.1 ns at pH 7 to 3.3 ns at pH 9.2. The lifetime data can be further interpreted as a decrease in the quenching of residues with a approximately 30 ps lifetime, and a concomitant increase in the longer lifetime components. This is consistent with interface tryptophans becoming exposed to solvent upon dissociation, and loss of quenching by intersubunit hemes. The overall results suggest that in the dodecamer, most of the tryptophans are located in a hydrophobic environment, not all of which are located at the subunit interface.

Absence of ligand binding-induced tertiary changes in the multimeric earthworm Lumbricus terrestris hemoglobin. A resonance Raman study.

In vertebrate hemoglobins, changes in protein tertiary structure induced by either ligand binding or changes in quaternary state are manifested at the heme as reflected in resonance Raman spectral changes involving the iron-proximal histidine stretching mode. No such changes are observed for Lumbricus terrestris hemoglobin. The iron-histidine stretching mode and the porphyrin breathing motion in the deoxy-, oxy-, or CO-photodissociated forms of Lumbricus hemoglobin and human hemoglobin A (pH 7.0 and 9.2, the latter to effect Lumbricus hemoglobin subunit dissociation) were studied using pulsed (10 ns) light at 435 nm. In contrast to that observed for hemoglobin A, a comparison of the spectra of the deoxy and photoproduct forms of Lumbricus hemoglobin reveal minimal differences in the region of the iron-histidine and the pi electron distribution in the heme moiety. The spectral frequencies are similar to that observed in R-state vertebrate hemoglobins. Such average behavior of the approximately 192 hemes present in Lumbricus hemoglobin is more analogous to the Raman spectral properties observed in myoglobin.

Unfolding and release of heme from human hemoglobins A, S and F.

1. Analysis of the Soret spectra of hemoglobins A, S and F has been used to determine the extent of heme exposure and release from these hemoglobins in the presence of several solvent perturbants. 2. Oxyhemoglobin S unfolding in the presence of either urea or propyl urea resulted in greater heme exposure and release than either oxyhemoglobins A or F. 3. Methemoglobin formation resulted in lower denaturation midpoints for each hemoglobin compared to the reduced oxyhemoglobin state; methemoglobin F had the lowest denaturation midpoint under isothermal denaturing conditions. 4. Rate of heme exposure was greater for oxyhemoglobin S than oxyhemoglobin A in the presence of 200 microM the anionic detergent sodium dodecyl sulfate. 5. Evidence for increased levels of heme release in hemoglobin S may be related to the greater tendency of sickled red cell membranes to undergo lipid oxidation.

Induced hemichrome formation of methemoglobins A, S and F by fatty acids, alkyl ureas and urea.

1. Spectral analysis of the Soret region (450-350 nm) has shown that saturated fatty acids, alkyl ureas and urea induce the conversion of methemoglobins A, S, and F to the hemichrome state. 2. In the presence of fatty acids (C8-C16), methemoglobin F is converted to the hemichrome state more readily than either methemoglobins A or S. 3. Using several alkyl ureas (methyl, ethyl, propyl, butyl), the extent of hemichrome formation was as follows: met Hb F > met Hb S > met Hb A. The ability of these compounds to induce hemichrome formation is related to their increasing hydrophobicity. 4. Conversion to the hemichrome state in the presence of urea (5M) led to the formation of molecular aggregates of hemoglobins S, F and A which may be initiated by subunit dissociation and conformational changes, coupled to increased globin-globin interactions. 5. Similar aggregation occurred for methemoglobin S in the presence of octanoic acid; no significant aggregation was evident for methemoglobin A after 10 hr of exposure to octanoic acid.

The differential effects of carbon monoxide and oxygen on the pressure dissociation of Lumbricus terrestris hemoglobin.

We have explored the subunit affinities of Lumbricus terrestris hemoglobin (LtHb) under a variety of conditions using high-pressure spectroscopy. While only small changes were observed for LtHb-oxy below 1.0 kbar, higher pressures resulted in a 1000 cm-1 red shift and 2-fold increase in fluorescence intensity with a concomitant 12-fold decrease in scattering intensity, all of which reached completion by approx. 2.2 kbar. In the presence of 1 M MgCl2 or at acidic pH (4.2), the curves shifted by 400 and 1000 bar corresponding to significant destabilization. At pH 9.1, the initial spectral parameters were almost equal to the final endpoints and were unaffected by pressure. While the pressure curve of the CO form was similar to the oxy form at pH 7.2, the midpoints of the other samples were shifted to higher pressures relative to their oxy counterpart, indicating tighter subunit contacts. This stabilization was unexpected based upon the sequence homology to vertebrate hemoglobins, and the minimal structural differences between these two liganded forms of human hemoglobin. These data indicate that the differences are the result of the additive nature of the interactions involved in subunit packing whose effects become significant in larger aggregates.

Spectroscopic analysis of the unfolding of transition metal-ion complexes of human lactoferrin and transferrin.

1. Human lactoferrin and transferrin are capable of binding several transition metal ions [Fe(III), Cu(II), Mn(III), Co(III)] into specific binding sites in the presence of bicarbonate. 2. Increased conformational stability and increased resistance to protein unfolding is observed for these metal-ion complexes compared to the apoprotein form of these proteins. 3. Mn(III)-lactoferrin and transferrin complexes exhibit steeper denaturation transitions than the Co(III) complexes of these proteins suggesting greater cooperativity in the unfolding process. 4. The incorporation of Fe(III) into the specific metal binding sites offers the greatest resistance to thermal unfolding when compared to the other transition metal ions studied. 5. Non-coincidence of unfolding transitions is observed, with fluorescence transition midpoints being lower than those determined by absorbance measurements. 6. Fully denatured proteins in the presence of urea and alkyl ureas exhibit fluorescence wavelength maxima at 355-356 nm indicative of tryptophan exposure upon protein unfolding.

Analysis of the acid and alkaline dissociation of earthworm hemoglobin, Lumbricus terrestris, by front-face fluorescence spectroscopy.

The steady-state fluorescence properties of the multisubunit hemoglobin isolated from the earthworm, Lumbricus terrestris, were studied by front-face fluorometry. Acid and alkaline dissociation of this high-molecular-weight hemoglobin were examined over the pH range 3.7-12.5 using different liganded states (oxy, CO, met). The relative intensity of the emission maximum at 320 nm (exc. 280 nm) is ligand-dependent increasing as follows: oxy less than deoxy less than CO less than met at pH 7.0. The intensity of the emission maximum of oxyhemoglobin at the alkaline acid end point, pH 10.5 (333 nm), is significantly greater than that observed at the acid end point, pH 4.18 (320 nm), suggesting different subunit dissociation. The spectra of oxyhemoglobin at pH 4.18 and the spectrum of carbonmonoxy hemoglobin at pH 7.0 in the presence of 1 M magnesium chloride were almost identical, indicating similar subunit dissociation. Difference spectrum (pH 9.0-7.2) of fluorescence emission (exc. 305) resulted in a maximum at 341 nm, indicative of tyrosinate formation. This suggests that tyrosine(s) may also be located at the subunit interface(s) of this hemoglobin. These studies indicate that several aromatic amino acid residues are associated with the critical sites of subunit interactions within this molecule. Analysis of the fluorescence spectra also suggests that the formation of different subunit species resulting from acid and alkaline dissociation cannot be ruled out.

Partial amino acid sequences of several globin chains from the sockeye salmon, Oncorhynchus nerka.

1. Partial amino acid sequences for several sockeye salmon hemoglobin beta-chains have been determined and compared to several other fish beta-chain sequences. 2. A 90% homology exists between the sockeye cathodal (C1) beta-chain and the trout Hb I beta-chain for residues 1-19. 3. The sockeye salmon anodal (A1-3) beta-chain is virtually identical to the trout HB IV beta-chain for the first 55 amino acid residues. 4. The alpha-chains of the sockeye salmon appear to be acetylated at the N-terminal position and about 0.6% of the sockeye hemoglobin is glycosylated.

Comparative studies of catalase and superoxide dismutase activity within salmon fish erythrocytes.

1. Superoxide dismutase isolated from erythrocytes of several species of salmon and the rainbow trout exhibited single electrophoretic bands of activity which migrated anodally similar to the human erythrocyte enzyme; two discrete bands were observed for the coho salmon. 2. No polymorphism was observed for 30 samples from sockeye salmon and six samples from king salmon. Only one sample of rainbow trout (one of 12) exhibited an electrophoretic mobility difference. 3. Catalase migration on starch-gel resembled the human enzyme's electrophoretic mobility for all salmon species and rainbow trout. Catalase activity of the sockeye salmon (2929 +/- 895 mumol min-1 gHb-1) was determined to be lower than human catalase activity. 4. All samples differed from the human enzymes in that they required the presence of a detergent, Triton X-100, for solubilization.

Anadara ovalis hemoglobins: distinct dissociation and ligand binding characteristics.

The red cells of the arcid clam Anadara ovalis contain two electrophoretically distinct hemoglobins: Hb Major (Hb Ma) and Hb Minor (Hb Mi). The major component consists of two electrophoretically indistinguishable tetramers each composed of two heterodimers; the minor hemoblogin is a homodimer whose subunits are different from the tetramer. Functionally, Hb Ma has a higher P50, exhibits a concentration dependent oxygen affinity, has significant ligand cooperativity (n = 2.0), lacks a Bohr effect and is unaffected by ATP. HB Mi has a P50 which is lower and independent of hemoglobin concentration, shows appreciable cooperativity (n = 1.4) and exhibits no heterotropic effects. Both Hb Ma and Mi are resistant to dissociation in the presence of 1.0 M NaI, NaCl and guanidine-HCl but dissociate to monomers when converted to the aquamet but not the cyanmet derivative. The dissociation is completely inhibited by mercaptoethanol. The large number of reactive -SH groups (10-13 per tetramer) suggests that the monomerization is mediated by intra-subunit disulfide bridge formation.

Unfolding of iron and copper complexes of human lactoferrin and transferrin.

1. Human lactoferrin and transferrin are capable of binding two iron or copper ions into specific binding sites in the presence of bicarbonate. 2. Urea and several alkyl ureas have been effective in unfolding these metal-protein complexes. 3. Biphasic transitions are observed for the unfolding of each of the metal complexes of these proteins as determined by direct visible spectroscopy suggesting the release of iron(III) and Cu(II) ions from both of these metal-binding proteins during the unfolding process. 4. Greater stabilization and increased resistance to protein unfolding is observed for all iron(III) complexes compared to Cu(II) complexes of lactoferrin and transferrin as determined by isothermal unfolding and thermal denaturation. 5. Relative stabilization of the different metal-protein complexes investigated within this study were determined to be as follows: Lf-Fe(III) greater than Lf-Cu(II); Tf-Fe(III) greater than Tf-Cu(II), and Lf-Fe(III) greater than Tf-Fe(III); Lf-Cu(II) greater than Tf-Cu(II).

Structural and functional studies of the king salmon, Oncorhynchus tshawytscha, hemoglobins.

Vertical starch-gel electrophoresis at pH 8.6 revealed extensive hemoglobin multiplicity with several distinct cathodal and anodal hemoglobin components. Anodal hemoglobin components are present throughout the life cycle of the king salmon. Additional cathodal components are found in the adult fish. Cathodal hemoglobin components exhibited a higher oxygen affinity (P50 = 10.2 mm at 13 degrees C, pH 7.3) than the anodal hemoglobin components (P50 = 21.8 mmHg at 13 degrees C). Oxygen binding of the anodal hemoglobins are sensitive to pH, temperature, organic phosphates (ATP and GTP), as well as, ionic strength; binding of oxygen to the cathodal hemoglobins is independent of pH and not affected by organic phosphates. Anodal hemoglobin components are less resistant to thermal denaturation over the pH 6.0 to 8.0 range. Isothermal urea denaturation of separated anodal and cathodal hemoglobin fractions of the king salmon indicate inherent differences in the stabilization energies of these hemoglobins. Autoxidation of these hemoglobins occurs around pH 7.0 and below, as well as, in the presence of increasing Cl- concentrations.