A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis.

The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.

Characterisation of radioactive waste products associated with plant decommissioning.

The inventory of radioactivity that must be considered in the decommissioning of a typical 1000 MWe Spanish pressurised water reactor (PWR) was investigated as part of a generic plant decommissioning study. Analyses based on DORT models (in both R-Z and R-theta geometries) were used with representative plant operating history and core power distribution data in defining the expected neutron environment in regions near the reactor core. The activation analyses were performed by multiplying the DORT scalar fluxes by energy-dependent reaction cross sections (based on ENDF/B-VI data) to generate reaction rates on a per atom basis. The results from the ORIGEN2 computer code were also used for determining the activities associated with certain nuclides where multi-group cross section data were not available. In addition to the bulk material activation of equipment and structures near the reactor, the activated corrosion-product (or 'crud') deposits on system and equipment surfaces were considered. The projected activities associated with these sources were primarily based on plant data and experience from operating PWR plants.

A distinct strategy to generate high-affinity peptide binders to receptor tyrosine kinases.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Mortality rates of interventional and surgical procedures performed in domestic juvenile farm pigs and Yucatan mini-pigs.

Perioperative and postoperative care are critical factors in cardiac catheterization and cardiothoracic surgical procedures. A retrospective analysis of mortality data in cardiovascular catheter and surgical studies performed in domestic juvenile swine (DJS) and Yucatan mini-swine (YMS) was conducted. A total of 529 animals in 35 studies were included in the analysis, which included six study categories: coronary stenting (Stent) and percutaneous transluminal coronary angioplasty (PTCA) alone; Stent and PTCA in combination with ionizing radiation (Stent/Rad, PTCA/Rad); myocardial ischemia (ISCH); and three non-ISCH surgical procedures grouped under "other surgeries" (Other Surg). Casualties were defined as animals that died spontaneously before the assigned termination date. The highest mortality rate occurred in the ISCH group (29.7% +/- 2.2%). Mortality of the Stent/Rad animals (26.1% +/- 6.3%) was significantly higher than those in the Stent and PTCA groups (12.1% +/- 3.1% and 7.9% +/- 3.2%; P< 0.05 for both). Similarly, mortality in the ISCH group was significantly higher than that in the Stent, PTCA, or Other Surg animals (29.7% +/- 2.2% versus 12.1% +/- 3.1%, 7.9% +/- 3.2%, and 3.0% +/- 3.0%, respectively; P< 0.05 for all comparisons). We did not observe differences between YMS and DJS. Most casualties in the ISCH group took place during weeks 1 (28.0% +/- 8.4%) and 4 (29.3% +/- 6.2%) after placement of the coronary ameroid constrictor. The majority of animals in the Stent/Rad and PTCA/Rad groups died within 1 week after the procedure (67.7% +/- 12.8% and 79.3% +/- 12.5%, respectively). We conclude that radiation therapy used in combination with stenting increases the mortality rate of this catheter-based procedure. Animals subjected to ISCH or a transcatheter procedure in combination with ionizing radiation should be monitored closely during the perioperative period to prevent unacceptably high mortality rates.

Alanine-scanning of the 50's loop in the Clostridium beijerinckii flavodoxin: evaluation of additivity and the importance of interactions provided by the main chain in the modulation of the oxidation-reduction potentials.

The four-residue reverse turn -Met56-Gly-Asp-Glu59- in the Clostridium beijerinckii flavodoxin provides the majority of the critical interactions with the isoalloxazine ring of the flavin mononucleotide (FMN) cofactor that contribute to the binding and the differential stabilization of its three redox states. Direct side chain contacts include the sulfur-ring interaction of Met56, which primarily influences the oxidized and hydroquinone states, and the hydrogen bond by Glu59 with the N3H, which directly (and indirectly through its "anchoring" function) influences all three states to various extents. Involving a novel redox-dependent conformational change, the hydrogen bond formed between the carbonyl group of Gly57 and the N5H of the reduced cofactor strongly influences the stability of the semiquinone state. In this study, the sequential elimination of all side chain interactions in various combinations through a systematic alanine-scanning mutagenesis approach was conducted to more completely understand the functional inter-relationships as well as any synergistic interactions that might occur within the loop. In general, additive effects for each side chain on the midpoint potentials for both couples were observed except for the hydroquinone state where some degree of nonadditivity was noted in multiple mutants involving Glu59. The study concluded with the generation of the triple mutant -Ala56-Gly-Ala-Ala59- in which all side chain interactions are removed. Gly57 was left unchanged because of its critical conformational contribution. Remarkably, this mutant retained the ability to bind the FMN and to thermodynamically stabilize the semiquinone state despite the absence of all side chain interactions. Collectively, these observations emphasize the overriding importance of the main chain interactions with the N5H of the FMN and the associated redox-dependent conformational change in this loop and leaves little doubt as to its role in the thermodynamic stabilization of the neutral semiquinone state of the FMN cofactor.

Role of hydrogen bonding interactions to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin.

The role of the hydrogen bonding interaction with the N(3)H of the flavin cofactor in the modulation of the redox properties of flavoproteins has not been extensively investigated. In the flavodoxin from Clostridium beijerinckii, the gamma-carboxylate group of glutamate-59 serves as a dual hydrogen bond acceptor with the N(3)H of flavin mononucleotide (FMN) cofactor and the amide hydrogen of the adjacent polypeptide backbone in all three oxidation states. This "bridging" interaction serves to anchor the FMN in the binding site, which, based on the E59Q mutant, indirectly affects the stability of the neutral flavin semiquinone by facilitating a strong and critical interaction at the FMN N(5)H [Bradley, L. H., and Swenson, R. P. (1999) Biochemistry 38, 12377-12386]. In this study, the specific role of the N(3)H interaction itself was investigated through the systematic replacement of Glu59 by aspartate, asparagine, and alanine in an effort to weaken, disrupt, and/or eliminate this interaction, respectively. Just as for the E59Q mutant, each replacement significantly weakened the binding of the cofactor, particularly for the semiquinone state, affecting the midpoint potentials of each one-electron couple in opposite directions. (1)H-(15)N HSQC nuclear magnetic resonance (NMR) spectroscopic studies revealed that not only was the N(3)H interaction weakened as anticipated, but so also was the hydrogen bonding interaction with the N(5)H. Using the temperature coefficients of the N(5)H to quantify and correct for changes in this interaction, the contribution of the N(3)H hydrogen bond to the binding of each redox state of the FMN was isolated and estimated. Based on this analysis, the N(3)H hydrogen bonding interaction appears to contribute primarily to the stability of the oxidized state (by as much as 2 kcal/mol) and to a lesser extent the reduced states. It is concluded that this interaction contributes only modestly (<45 mV) to the modulation of the midpoint potential for each redox couple in the flavodoxin. These conclusions are generally consistent with ab initio calculations and model studies on the non-protein-bound cofactor.

Evaluation of the hydrogen bonding interactions and their effects on the oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin.

The oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin are substantially different from those of the flavin mononucleotide (FMN) containing native protein, with the midpoint potential for the semiquinone-hydroquinone couple for the riboflavin complex being 180 mV less negative. This increase has been attributed to the absence in the riboflavin complex of unfavorable electrostatic effects of the dianionic 5'-phosphate of the FMN on the stability of the flavin hydroquinone anion. In this study, 15N and 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopic studies demonstrate that when bound to the flavodoxin, (1) the N1 of the riboflavin hydroquinone remains anionic at pH 7.0 so the protonation of the hydroquinone is not responsible for this increase, (2) the N5 position is much more exposed and may be hydrogen bonded to solvent, and (3) that while the hydrogen bonding interaction at the N3H appears stronger, that at the N5H in the reduced riboflavin is substantially weaker than for the native FMN complex. Thus, the higher reduction potential of the riboflavin complex is primarily the consequence of altered interactions with the flavin ring that affect hydrogen bonding with the N5H that disproportionately destabilize the semiquinone state of the riboflavin rather than through the absence of the electrostatic effects of the 5'-phosphate on the hydroquinone state.

Different underlying mechanisms for deficits in concept formation in dementia.

We investigated the different mechanisms that may underlie deficits in verbal concept formation among patients with Alzheimer's disease (AD) and ischaemic vascular dementia (IVD) associated with periventricular and deep white matter alterations. Concept formation was assessed with the WAIS-R Similarities subtest (SIM). Two types of errors were re-coded from the 0-point responses as scored by the WAIS-R manual. In set errors (e.g., dog-lion "they're alive") were coded when patients reported a very vague superordinate concept for the word pair. Out of set responses (e.g., dog-lion "the lion roars and the dog barks") were coded when a response was clearly out of mental set, i.e., when participants were unable to provide a superordinate concept for the word pair. Between-group comparisons demonstrated no difference in SIM test performance according to the scoring system described in the WAIS-R manual. Nonetheless, AD patients produced a greater proportion of in set errors, while IVD patients produced a greater proportion of out of set errors. Out of set errors were highly associated with measures of executive function, while in set errors were associated with measures related to delayed recognition memory and semantic intrusion errors. We conclude that the underlying deficits that contribute to poor concept formation differ between AD and IVD patients. In IVD impaired concept formation is related to deficits in the executive systems necessary to monitor responses and sustain mental set. In AD, by contrast, the deficit appears to be secondary to impaired verbal response selection.

Neuropsychological profiles associated with subcortical white matter alterations and Parkinson's disease: implications for the diagnosis of dementia.

Despite the emergence of a number of new classification systems, the diagnosis of cerebrovascular dementia remains controversial. Also controversial is the significance of periventricular and deep white matter alterations (WMA) as seen on magnetic resonance imaging (MRI). To further clarify this issue, MRI scans were used to regroup patients clinically diagnosed with Alzheimer's disease (AD) or subcortical ischemic vascular dementia (IVD) into cohorts presenting with either little versus significant WMA on MRI. These two groups were then compared to demented patients diagnosed with idiopathic Parkinson's disease (PD) using a comprehensive neuropsychological protocol. Neuropsychological assessment failed to distinguish between patients with PD and significant WMA. By contrast, both of these patient groups exhibited disproportionate impairment on tests of executive systems functioning, whereas patients with little WMA showed greater impairment on tests of declarative memory and semantic knowledge. These findings constitute further evidence that the pattern of cognitive impairment associated with significant WMA is distinctly different when compared to AD. These results are discussed within the context of a growing body of literature suggesting that elements of the underlying neuropathologies in AD and IVD are linked. Implications for the diagnosis of dementia are also discussed.

Conformational energetics of a reverse turn in the Clostridium beijerinckii flavodoxin is directly coupled to the modulation of its oxidation-reduction potentials.

A surface loop in the flavodoxin from Clostridium beijerinckii comprised of residues -Met(56)-Gly-Asp-Glu(59)- forms a four-residue reverse turn which undergoes a conversion from a mix of cis/trans peptide configurations that approximate a type II configuration in the oxidized state to a type II' turn upon reduction of the bound flavin mononucleotide (FMN) cofactor. This change results in the formation of a new hydrogen bond between the N(5)H of the reduced cofactor and the carbonyl group of Gly57 of the central peptide bond of the turn, an interaction that is thought to contribute to the modulation of the oxidation-reduction potentials of the cofactor [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. In this study, the direct linkage of the conformational energetics of this turn to the stabilization of the FMN semiquinone was established by systematically replacing the second and third residues of the turn (Gly57 and Asp58) with the -Gly-Gly-, -Gly-Ala-, -Ala-Gly-, and -Ala-Ala- dipeptidyl sequences. On the basis of published position specific preferences for residues with side chains (mimicked by Ala) and glycine, a strong correlation was observed between E(ox/sq) and the calculated free-energy differences between the type II and type II' conformations of each of these sequence combinations. The -Ala-Gly- sequence, which favors the type II turn configuration primarily adopted in the oxidized state, displays a E(ox/sq) value that is about 150 mV more negative than that for the wild-type-like -Gly-Ala- sequence, which prefers the type II' conformation observed in the reduced states. The -Gly-Gly- and -Ala-Ala- mutants exhibit intermediate E(ox/sq) values consistent with their ambivalent turn preferences. The potential changes are primarily the result of alterations in the stability of the semiquinone state. These results provide more conclusive evidence for the crucial role of this conformational change in the modulation of the redox potentials of this flavodoxin. Furthermore, this study establishes a direct association between the conformational energetics of the protein, induced in this case by the sequence specificity of a beta-turn, and the differential thermodynamic stabilization of specific redox states of the cofactor, demonstrating another means by which flavoproteins can modulate the redox potentials of the bound cofactor.

Effect of dietary supplements containing antioxidants on attenuation of muscle damage in exercising sled dogs.

OBJECTIVE: To determine whether dietary antioxidants would attenuate exercise-induced increases in plasma creatine kinase (CK) activity in sled dogs. ANIMALS: 41 trained adult sled dogs. PROCEDURE: Dogs, randomly assigned to 2 groups, received the same base diet throughout the study. After 8 weeks on that diet, 1 group (21 dogs) received a daily supplement containing vitamins E (457 U) and C (706 mg) and beta-carotene (5.1 mg), and a control group (20 dogs) received a supplement containing minimal amounts of antioxidants. After 3 weeks, both groups performed identical endurance exercise on each of 3 days. Blood samples were collected before and 3 weeks after addition of supplements and after each day of exercise. Plasma was analyzed for vitamins E and C, retinol, uric acid, triglyceride, and cholesterol concentrations, total antioxidant status (TAS), and CK activity. RESULTS: Feeding supplements containing antioxidants caused a significant increase in vitamin E concentration but did not change retinol or vitamin C concentrations orTAS. Exercise caused significantly higher CK activity, but did not cause a significant difference in CK activity between groups. Exercise was associated with significantly lower vitamin E, retinol, and cholesterol concentrations and TAS but significantly higher vitamin C, triglyceride, and uric acid concentrations in both groups. CONCLUSIONS AND CLINICAL RELEVANCE: Use of supplements containing the doses of antioxidants used here failed to attenuate exercise-induced increases in CK activity. Muscle damage in sled dogs, as measured by plasma CK activity, may be caused by a mechanism other than oxidant stress.

Shoulder numbness in a patient with suprascapular nerve entrapment syndrome: cutaneous branch of the suprascapular nerve: case report.

OBJECTIVE AND IMPORTANCE: The ability to diagnose peripheral nerve disorders is dependent on knowledge of the anatomic course and function of the nerves in question. The classic teaching regarding the suprascapular nerve (SScN) is that it has no cutaneous branches, despite the fact that a cutaneous branch was first reported in the anatomic literature 20 years ago. CLINICAL PRESENTATION: We describe a case of a 35-year-old male patient who presented with right shoulder pain and atrophy and weakness of the right supra- and infraspinatus muscles. During the examination, he was also noted to have an area of numbness involving the right upper lateral shoulder region. Electrical study results were consistent with SScN entrapment at the suprascapular notch. INTERVENTION: The patient underwent surgical decompression 7 months after the onset of his symptoms. The patient noted resolution of his shoulder pain immediately after the procedure, and his shoulder sensory disturbance had improved by 2 weeks. At 9 months after surgery, he remained pain-free, his shoulder sensation was normal, and his motor abnormalities had improved significantly. CONCLUSION: This case provides clinical evidence for the presence of a cutaneous branch of the SScN, as described in cadaveric studies. Although shoulder numbness demands a search for alternative diagnoses, it does not necessarily exclude the diagnosis of SScN entrapment.

Effects of dietary antioxidant supplementation on oxidative damage and resistance to oxidative damage during prolonged exercise in sled dogs.

OBJECTIVES: To determine effects of dietary antioxidant supplementation on plasma concentrations of antioxidants, exercise-induced oxidative damage, and resistance to oxidative damage during exercise in Alaskan sled dogs. ANIMALS: 62 Alaskan sled dogs. PROCEDURE: Dogs were matched for age, sex, and ability and assigned to 1 of 3 groups: sedentary and nonsupplemented (control [C]; n = 21), exercised and supplemented (S; 22), and exercised and nonsupplemented (N; 19). Dogs in group S were given 400 units of alpha-tocopherol acetate, 3 mg of beta-carotene, and 20 mg of lutein orally per day for 1 month, then dogs in groups S and N completed 3 days of exercise. Blood samples were collected before and after 1 and 3 days of exercise and after 3 days of rest. Plasma antioxidant concentrations were determined, and oxidative damage to DNA (plasma 7,8 dihydro-8-oxo-2'deoxyguanosine [8-oxodG] concentration) and membrane lipids (plasma hydroperoxide concentration) and resistance of plasma lipoproteins to oxidation were assessed. RESULTS: Supplementation increased plasma concentrations of alpha-tocopherol, beta-carotene, and lutein. Plasma concentration of alpha-tocopherol increased and concentration of lutein decreased in group S with exercise. Concentration of 8-oxodG decreased in group S but increased in group N during and after exercise. Lag time of in vitro oxidation of lipoprotein particles increased with exercise in group S only. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary supplementation with antioxidants resulted in increased plasma concentrations of antioxidants. Moreover, supplementation decreased DNA oxidation and increased resistance of lipoprotein particles to in vitro oxidation. Antioxidant supplementation of sled dogs may attenuate exercise-induced oxidative damage.

Oxidant stress in sled dogs subjected to repetitive endurance exercise.

OBJECTIVE: To determine whether repetitive endurance exercise in sled dogs was associated with substantial lipid peroxidation, decreases in antioxidant capacity of the serum, and skeletal muscle damage. ANIMALS: 24 lightly trained sled dogs. PROCEDURE: 16 dogs completed a 58-km run on each of 3 consecutive days; the other 8 dogs (control) did not exercise during the study. Blood samples were collected before the first exercise run and after the first and third exercise runs. Plasma isoprostane and serum vitamin E concentrations, total antioxidant status of plasma, and serum creatine kinase activity were measured. RESULTS: Plasma isoprostane concentrations in dogs in the exercise group were significantly increased after the first exercise run and further significantly increased after the third exercise run. Serum vitamin E concentration was significantly decreased after the first exercise run in dogs in the exercise group, and this change persisted after the third exercise run. There was a significant linear relationship between plasma isoprostane concentration and the logarithm of serum creatine kinase activity (adjusted ? = 0.84). CONCLUSIONS AND CLINICAL RELEVANCE: Results demonstrate that repetitive endurance exercise in dogs is associated with lipid peroxidation and a reduction in plasma antioxidant concentrations. We interpret these results as indicating that the antioxidant mechanisms of minimally trained dogs may, in some instances, be inadequate to meet the antioxidant requirements of repetitive endurance exercise.

Spontaneous order, autocatakinetic closure, and the development of space-time

Over its 4.6 billion year history, the time-dependent behavior of planet Earth, from the origin and emergence of life to the explosive globalization of human culture today, shows the progressive and accelerating production of increasingly more highly ordered dynamic states. Understanding our place as both productions and producers in this rapidly accelerating global becoming is a requisite step to the meaningful grounding of virtually every other discipline, most particularly those disciplines relating to the endeavors and activities of humans themselves. Recent advances in the study of spontaneous ordering provide both a minimal ontological framework required for causally addressing such systems, and the nomological basis for understanding the ubiquitous or universal generic nature of such ordering itself. This paper briefly outlines the main points.

Discovery of a series of cyclohexylethylamine-containing protein farnesyltransferase inhibitors exhibiting potent cellular activity.

Synthesis of a library of secondary benzylic amines based on the Sebti-Hamilton type peptidomimetic farnesyltransferase (FTase) inhibitor FTI-276 (1) led to the identification of 6 as a potent enzyme inhibitor (IC(50) of 8 nM) which lacked the problematic thiol residue which had been a common theme in many of the more important FTase inhibitors reported to date. It has previously been disclosed that addition of o-tolyl substitution to FTase inhibitors of the general description 2 had a salutary effect on both FTase inhibition and inhibition of Ras prenylation in whole cells. Combination of these two observations led us to synthesize 7, a potent FTase inhibitor which displayed an IC(50) of 0.16 nM for in vitro inhibition of FTase and an EC(50) of 190 nM for inhibition of whole cell Ras prenylation. Modification of 7 by classical medicinal chemistry led to the discovery of a series of potent FTase inhibitors, culminating in the identification of 25 which exhibited an IC(50) of 0.20 nM and an EC(50) of 4.4 nM. In vivo tests in a nude mouse xenograft model of human pancreatic cancer (MiaPaCa cells) showed that oral dosing of 25 gave rise to impressive attenuation of the growth of this aggressive tumor cell line.

Role of glutamate-59 hydrogen bonded to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin. Glutamate-59 is not responsible for the pH dependency but contributes to the stabilization of the flavin semiquinone.

The midpoint potentials for both redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor in the flavodoxin are known to be pH dependent. While the pH dependency for the oxidized-semiquinone (ox/sq) couple is consistent with the formation of the blue neutral form of the flavin semiquinone, that of the semiquinone-hydroquinone (sq/hq) couple is more enigmatic. The apparent pK(a) of 6.7 for this couple in the flavodoxin from Clostridium beijerinckii has been attributed to the ionization of the FMN(HQ); however, nuclear magnetic resonance data strongly suggest the FMN(HQ) remains anionic over the entire pH range testable. As an alternative explanation, a specific glutamate residue (Glu59 in this flavodoxin), which is hydrogen-bonded to N(3)H of the FMN, has been postulated to be the primary redox-linked proton acceptor responsible for the pH effect in some flavodoxins. This model was directly tested in this study by permanently neutralizing Glu59 by its replacement with glutamine. This conservative substitution resulted in an increase of 86 mV (at pH 7) in midpoint potential of the sq/hq couple; however, the pH dependency of this couple was not altered. Thus, the redox-linked protonation of Glu59 clearly cannot be responsible for this effect as proposed. The pH dependency of the ox/sq couple was also similar to wild type, but the midpoint potential has decreased by 65 mV (pH 7). The K(d) values for the oxidized, semiquinone, and hydroquinone complexes increased by 43-, 590-, and 20-fold, respectively, relative to the wild type. Thus, the Glu59 to glutamine substitution substantially effects the stability of the semiquinone but, on a relative basis, slightly favors the formation of the hydroquinone. On the basis of (1)H-(15)N HSQC nuclear magnetic resonance spectroscopic studies, the increased temperature coefficients for the protons on N(3) and N(5) of the reduced FMN in E59Q suggest that the hydrogen-bonding interactions at these positions are significantly weakened in this mutant. The increase for N(5)H correlates with the reduced stability of the FMN(SQ) and the more negative midpoint potential for the ox/sq couple. On the basis of the X-ray structure, an "anchoring" role is proposed for the side chain carboxylate of Glu59 that stabilizes the structure of the 50's loop in such a way so as to promote the crucial hydrogen-bonding interaction that stabilizes the flavin semiquinone, contributing to the low potential of this flavodoxin.

Thermodynamics in the present progressive mode and its role in the context of the origin of life.

The origin and evolution of biological organizations proceeding on Earth are put in a nonequilibrium thermodynamic framework within a cosmological context. The dynamic process responsible for chemical evolution leading to the origin of biological being depends upon consumer-dominating thermodynamics, in which the heat sink is taken to be active in extracting heat energy from a body at a higher temperature. Consumer-dominating thermodynamics follows from the fact that when a small hot body contacts a cold heat sink, it decreases the temperature at the possible fastest rate. The fastest temperature drop, when applied to chemical products being synthesized through the energy supplied from an external heat source, is selective in keeping only those products that can decrease the temperature at the fastest rate among the available alternatives. Synthesis of small organic molecules in the small ice grains in interstellar diffuse clouds irradiated by ultraviolet radiation is a representative case of consumer-dominating thermodynamics, in which diffuse clouds serve as cold heat sinks in the cosmological context. Another case of consumer-dominating thermodynamics predominant on Earth especially in the perspective of the origin and evolution of life is with submarine hydrothermal vents, in which the surrounding cold seawater constantly serves as the cold heat sink.

The midpoint potentials for the oxidized-semiquinone couple for Gly57 mutants of the Clostridium beijerinckii flavodoxin correlate with changes in the hydrogen-bonding interaction with the proton on N(5) of the reduced flavin mononucleotide cofactor as measured by NMR chemical shift temperature dependencies.

In the Clostridium beijerinckii flavodoxin, the reduction of the flavin mononucleotide (FMN) cofactor is accompanied by a local conformation change in which the Gly57-Asp58 peptide bond "flips" from primarily the unusual cis O-down conformation in the oxidized state to the trans O-up conformation such that a new hydrogen bond can be formed between the carbonyl group of Gly57 and the proton on N(5) of the neutral FMN semiquinone radical [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. This interaction is thought to contribute to the relative stabilization of the flavin semiquinone and may be at least partially responsible for the substantial separation of the midpoint potentials of the two one-electron reduction steps. Through a series of amino acid substitutions, the above cited study demonstrated the critical role of the often conserved glycine residue in this process. However, it has not been directly established experimentally as to whether these substitutions brought about the changes in the midpoint potentials by altering the strength of this hydrogen-bonding interaction as proposed. In this study, the relative strengths of the FMN N(5)H.O57 hydrogen bond in wild type and the G57A, G57N, and G57T mutants were evaluated by measuring the temperature dependency of the chemical shift for the proton on N(5) of the fully reduced cofactor by 1H-15N HSQC nuclear magnetic resonance spectroscopy. Based on the established correlation between the temperature coefficient of amide protons and the strength of hydrogen bonding in small peptides, the apparent strength of the N(5)H.O57 interaction was found to depend on the properties of the side chain at position 57. The glycine residue found in the wild-type flavodoxin appears to provide the strongest interaction while the beta-branched side chain in the G57T mutant provides the weakest. A good correlation was noted between the temperature coefficients of N(5)H and the one-electron reduction potential for the ox/sq couple as well as the binding free energy of the FMN semiquinone in this group of mutants. These results provide more direct quantitative evidence that support the previous hypothesis that this conformation change and the associated formation of the hydrogen bonding interaction with N(5)H of the reduced FMN represent an important means of stabilizing the neutral semiquinone and in modulating the oxidation-reduction potentials of the flavin cofactor in this and perhaps other flavodoxins.