Plasma volume expansion with solutions of hemoglobin, albumin, and Ringer lactate in sheep.
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We have measured plasma volume expansion (Evans blue and hematocrit changes) and hemodynamic responses in conscious hemorrhaged and normovolemic splenectomized sheep after a 30-min infusion of either 20 ml/kg of diaspirin cross-linked hemoglobin (DCLHb), 20 ml/kg of human albumin (Alb), or 60 ml/kg of a solution of Ringer lactate (RL). All regimens expanded blood volume and increased blood pressure and cardiac output after hemorrhage. However, only 15 +/- 3% of the infused volume of RL was evident as intravascular expansion 10-min postinfusion, compared with 67 +/- 16% and 139 +/- 139% for Alb and DCLHb, respectively. DCLHb infusions were associated with higher blood pressures and lower cardiac outputs compared with RL and Alb infusions, but the increased oxygen content of blood with DCLHb resulted in systemic delivery of oxygen similar to that of the other infusions. These differences in hemodynamics and vascular volume continued for 6 h, and at 24 h vascular volume and all hemodynamics were similar in all three groups. The better volume expansion with DCLHb may be due to greater mobilization of endogenous interstitial protein or reduced transcapillary loss as total intravascular endogenous plasma protein increased after infusion of DCLHb, whereas there was an apparent loss of endogenous intravascular protein after infusions of Alb and RL. Vasoconstriction by DCLHb is one mechanism that could lower blood-to-tissue transport of fluid and protein. In addition to its oxygen-carrying capacity and vasoactivity, DCLHb is associated with volume expansion properties out of proportion to its colloid osmotic pressure. (+info)
Osmolality: a physiological long-term regulator of lumbar sympathetic nerve activity and arterial pressure.
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Acute infusion of hypertonic fluid increases mean arterial pressure (MAP) in part by elevating nonrenal sympathetic activity. However, it is not known whether chronic, physiological increases in osmolality also increase sympathetic activity. To test this hypothesis, MAP, heart rate (HR), and lumbar sympathetic nerve activity (LSNA) were measured in conscious, 48-h water-deprived rats (WD) during a progressive reduction in osmolality produced by a 2-h systemic infusion (0.12 ml/min) of 5% dextrose in water (5DW). Water deprivation significantly increased osmolality (308 +/- 2 vs. 290 +/- 2 mosmol/kgH2O, P < 0.001), HR (453 +/- 7 vs. 421 +/- 10 beats/min, P < 0.05), and LSNA (63.5 +/- 1.8 vs. 51.9 +/- 3.8% baroreflex maximum, P < 0.01). Two hours of 5DW infusion reduced osmolality (-15 +/- 5 mosmol/kgH2O), LSNA (-23 +/- 3% baseline), and MAP (-10 +/- 1 mmHg). To evaluate the role of vasopressin in these changes, rats were pretreated with a V1-vasopressin receptor antagonist. The antagonist lowered MAP (-5 +/- 1 mmHg) and elevated HR (32 +/- 7 beats/min) and LSNA (11 +/- 3% baseline) in WD (P < 0. 05), but not in water-replete, rats. 5DW infusion had a similar cumulative effect on all variables in V1-blocked WD rats, but had no effect in water-replete rats. Infusion of the same volume of normal saline in WD rats did not change osmolality, LSNA or MAP. Together these data indicate that, in dehydrated rats, vasopressin supports MAP and suppresses LSNA and HR and that physiological changes in osmolality directly influence sympathetic activity and blood pressure independently of changes in vasopressin and blood volume. (+info)
Effect of ionic strength on the interfacial properties of cytochrome c.
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The surface tension behaviour of oxidised cytochrome c (cyt c) solution was characterised at various pH and ionic strength at the air/water interface. The pendant drop method employing digital image analysis of the drop shape was applied to the measurement of the surface tension. The adsorption properties of cyt c were utilised to study the protein conformation change effected by acidification and ionic strength. At high ionic strength, the saturated steady-state surface tension shows a cooperative change centred around 3.6 induced by a decrease in pH. Using spectroscopic experiments, the apparent pK of the acid-induced transition of horse cyt c from the native to the molten globular state is equal to 3.5. This fact indicates that the saturated steady-state surface tension is a parameter which might be used to monitor conformation changes of cyt c. (+info)
Solution structure of substrate-based ligands when bound to hepatitis C virus NS3 protease domain.
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The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors. (+info)
Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity.
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Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element. (+info)
Binding of a dimeric derivative of vancomycin to L-Lys-D-Ala-D-lactate in solution and at a surface.
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BACKGROUND: The emergence of bacteria that are resistant to vancomycin (V), a glycopeptide antibiotic, results from the replacement of the carboxy-terminal D-Ala-D-Ala of bacterial cell wall precursors by D-Ala-D-lactate. Recently, it has been demonstrated that covalent dimeric variants of V are active against vancomycin-resistant enterococci (VRE). To study the contribution of divalency to the activities of these variants, we modeled the interactions of V and a dimeric V with L-Lys-D-Ala-D-lactate, an analog of the cell-wall precursors of the vancomycin-resistant bacteria. RESULTS: A dimeric derivative of V (V-Rd-V) was found to be much more effective than V in inhibiting the growth of VRE. The interactions of V and V-Rd-V with a monomeric lactate ligand - diacetyl-L-Lys-D-Ala-D-lactate (Ac2KDADLac) - and a dimeric derivative of L-Lys-D-Ala-D-lactate (Lac-R'd-Lac) in solution have been examined using isothermal titration calorimetry and UV spectroscopy titrations; the results reveal that V-Rd-V binds Lac-R'd-Lac approximately 40 times more tightly than V binds Ac2KDADLac. Binding of V and of V-Rd-V to Nalpha-Ac-L-Lys-D-Ala-D-lactate presented on the surface of mixed self-assembled monolayers (SAMs) of alkanethiolates on gold indicates that the apparent off-rate for dissociation of V-Rd-V from the surface is much slower than that of V from the same surface. CONCLUSIONS: The results are compatible with the hypothesis that divalency is responsible for tight binding, which correlates with small values of minimum inhibitory concentrations of V and V-Rd-V. (+info)
Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes.
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EmrE is a 12-kDa Escherichia coli multidrug transporter that confers resistance to a wide variety of toxic reagents by actively removing them in exchange for hydrogen ions. The three native Cys residues in EmrE are inaccessible to N-ethylmaleimide (NEM) and a series of other sulfhydryls. In addition, each of the three residues can be replaced with Ser without significant loss of activity. A protein without all the three Cys residues (Cys-less) has been generated and shown to be functional. Using this Cys-less protein, we have now generated a series of 48 single Cys replacements throughout the protein. The majority of them (43) show transport activity as judged from the ability of the mutant proteins to confer resistance against toxic compounds and from in vitro analysis of their activity in proteoliposomes. Here we describe the use of these mutants to study the accessibility to NEM, a membrane permeant sulfhydryl reagent. The study has been done systematically so that in one transmembrane segment (TMS2) each single residue was replaced. In each of the other three transmembrane segments, at least four residues covering one turn of the helix were replaced. The results show that although the residues in putative hydrophilic loops readily react with NEM, none of the residues in putative transmembrane domains are accessible to the reagent. The results imply very tight packing of the protein without any continuous aqueous domain. Based on the findings described in this work, we conclude that in EmrE the substrates are translocated through a hydrophobic pathway. (+info)
Structure of the subunit c oligomer in the F1Fo ATP synthase: model derived from solution structure of the monomer and cross-linking in the native enzyme.
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The structure of the subunit c oligomer of the H+-transporting ATP synthase of Escherichia coli has been modeled by molecular dynamics and energy minimization calculations from the solution structure of monomeric subunit c and 21 intersubunit distance constraints derived from cross-linking of subunits. Subunit c folds in a hairpin-like structure with two transmembrane helices. In the c12 oligomer model, the subunits pack to form a compact hollow cylinder with an outer diameter of 55-60 A and an inner space with a minimal diameter of 11-12 A. Phospholipids are presumed to pack in the inner space in the native membrane. The transmembrane helices pack in two concentric rings with helix 1 inside and helix 2 outside. The calculations strongly favor this structure versus a model with helix 2 inside and helix 1 outside. Asp-61, the H+-transporting residue, packs toward the center of the four transmembrane helices of two interacting subunits. From this position at the front face of one subunit, the Asp-61 carboxylate lies proximal to side chains of Ala-24, Ile-28, and Ala-62, projecting from the back face of a second subunit. These interactions were predicted from previous mutational analyses. The packing supports the suggestion that a c-c dimer is the functional unit. The positioning of the Asp-61 carboxyl in the center of the interacting transmembrane helices, rather than at the periphery of the cylinder, has important implications regarding possible mechanisms of H+-transport-driven rotation of the c oligomer during ATP synthesis. (+info)