Safety and efficacy of oral low-volume sodium phosphate bowel preparation for colonoscopy in dogs.

BACKGROUND: Sodium phosphate (NaP) is a low-volume, hyperosmolar laxative that is an effective bowel-cleansing agent in humans. HYPOTHESIS: NaP will be as safe and efficacious as polyethylene glycol (PEG) bowel preparation for colonoscopy in dogs. ANIMALS: Eight purpose-bred healthy dogs. METHODS: In phase I, standard (NaP and enemas; NaP(1)) and control preparations (PEG and enemas) were compared in a crossover design to determine the safety and efficacy of NaP. Serial clinical and serum analytical evaluations were used to determine the safety of NaP. In phase II, the efficacy of the standard NaP preparation was compared with 3 other NaP variations, which excluded enema or included bisacodyl, with or without enemas in a crossover design. An observer blinded to the bowel preparation assigned a score of 1-4 (1=clean colon; 4=unacceptable colon cleansing preventing adequate endoscopic evaluation) to each of 5 regions of the colon. RESULTS: The mean total colon cleansing score (TCS), defined as the sum of scores from each region, of the control (9.4) was less than NaP(1) (13.6) (P < 0.05). There were no significant differences in regional or TCS for the remaining 4 NaP protocols. NaP(1) resulted in moderate, but clinically occult, hyperphosphatemia and hypocalcemia, which resolved within 24 hours. CONCLUSIONS AND CLINICAL IMPORTANCE: Despite the safety and ease of administration of the NaP preparations, the NaP bowel-cleansing preparations used in this study cannot be recommended for use because of the inadequate quality of bowel preparation compared with the protocol using PEG-containing fluids.

In vitro interaction of the Escherichia coli cyclic AMP receptor protein with the lactose repressor.

Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and lactose repressor associate to form a 2:1 complex in vitro. This is, to our knowledge, the first demonstration of a direct interaction of these proteins in the absence of DNA. No 1:1 complex was detected over a wide range of CAP concentrations, suggesting that binding is highly cooperative. Complex formation is stimulated by cAMP, with a net uptake of 1 equivalent of cAMP per molecule of CAP bound. Substitution of the dimeric lacI-18 mutant repressor for tetrameric wild-type repressor completely eliminates detectable binding. We therefore propose that CAP binds the cleft between dimeric units in the repressor tetramer. CAP-lac repressor interactions may play important roles in regulatory events that take place at overlapping CAP and repressor binding sites in the lactose promoter.

Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide.

Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.

Brain abnormalities in Gulf War syndrome: evaluation with 1H MR spectroscopy.

PURPOSE: To test for neuronal brain damage in the basal ganglia and brainstem in Gulf War veterans by using magnetic resonance (MR) spectroscopy. MATERIALS AND METHODS: Twenty-two Gulf War veterans with one of three factor analysis-derived syndromes (case patients); 18 well veterans matched for age, sex, and education level (control subjects); and six Gulf War veterans with syndrome 2 from a different population (replication sample) underwent long echo time (272 msec) proton (hydrogen 1) MR spectroscopy on a 4 x 2 x 2-cm voxel in the basal ganglia bilaterally and a 2 x 2 x 2-cm voxel in the pons. Syndromes 1-3 are described as "impaired cognition," "confusion-ataxia," and "central pain," respectively. RESULTS: The N-acetylaspartate-to-creatine (NAA/Cr) ratio, which reflects functional neuronal mass, was significantly lower in the basal ganglia and brainstem of Gulf War veterans with the three syndromes than in those structures of the control subjects (P =.007). The finding was corroborated in the replication sample (P =.002). Veterans with syndrome 2 (the most severe clinically) had evidence of decreased NAA/Cr in both the basal ganglia and the brainstem; those with syndrome 1, in the basal ganglia only; and those with syndrome 3, in the brainstem only. CONCLUSION: Veterans with different Gulf War syndromes have biochemical evidence of neuronal damage in different distributions in the basal ganglia and brainstem.

Participation of the amino-terminal domain in the self-association of the full-length yeast TATA binding protein.

The association of monomeric TATA binding protein with promoter DNA is an essential first step in many current models of eukaryotic transcription initiation. This step is followed by others in which additional transcription factors, and finally RNA polymerase, assemble at the promoter. Here we characterize the quaternary interactions of the Saccharomyces cerevisiae TATA-binding protein (yTBP), in the absence of other proteins or DNA. The data reveal a robust pattern in which yTBP monomers equilibrate with tetramers and octamers over a broad span of temperatures (4 degrees C

The TATA-binding protein from Saccharomyces cerevisiae oligomerizes in solution at micromolar concentrations to form tetramers and octamers.

Equilibrium analytical ultracentrifugation has been used to determine the stoichiometry and energetics of the self-assembly of the TATA-binding protein of Saccharomyces cerevisiae at 30 degreesC, in buffers ranging in salt concentration from 60 mM KCl to 1 M KCl. The data are consistent with a sequential association model in which monomers are in equilibrium with tetramers and octamers at protein concentrations above 2.6 microM. Association is highly cooperative, with octamer formation favored by approximately 7 kcal/mol over tetramers. At high [KCl], the concentration of tetramers becomes negligible and the data are best described by a monomer-octamer reaction mechanism. The equilibrium association constants for both monomer <--> tetramer and tetramer <--> octamer reactions change with [KCl] in a biphasic manner, decreasing with increasing [KCl] from 60 mM to 300 mM, and increasing with increasing [KCl] from 300 mM to 1 M. At low [KCl], approximately 3 mole equivalents of ions are released at each association step, while at high [KCl], approximately 3 mole equivalents of ions are taken up at each association step. These results suggest that there is a salt concentration-dependent change in the assembly mechanism, and that the mechanistic switch takes place near 300 mM KCl. The possibility that this self-association reaction may play a role in the activity of the TATA-binding protein in vivo is discussed.

Electrophoretic analysis of multiple protein-DNA interactions.

Under favorable conditions, native gel electrophoresis allows the resolution of protein-DNA complexes that differ in stoichiometry, identities of occupied DNA sequences (configuration), and macromolecular conformation. This technique provides a unique opportunity to analyze, in thermodynamic terms, the molecular interactions that govern the equilibrium distributions of species in protein-DNA mixtures. Here we describe a general theoretical approach to the analysis of electrophoretic band intensities, and provide examples of its application to the analysis of several interacting systems.

Role of macromolecular hydration in the binding of the Escherichia coli cyclic AMP receptor to DNA.

The osmotic stress technique was used to measure the changes in macromolecular hydration that accompany binding of the Escherichia coli CAP protein to its transcription-regulatory site (C1) in the lactose promoter and that accompany the transfer of CAP from site C1 to nonspecific genomic DNA. Formation of the C1 complex is accompanied by the net release of 79 +/- 11 water molecules. If all water molecules were released from macromolecular surfaces, this result would be consistent with a net reduction of solvent-accessible surface area of 711 +/- 189 A2. This area is only slightly smaller than the solvent-inaccessible macromolecular interface in crystalline CAP-DNA complexes. The transfer of CAP from site C1 to nonspecific sites is accompanied by the net uptake of 56 +/- 10 water molecules. Taken with the water stoichiometry of sequence-specific binding, this value implies that formation of a nonspecific complex is accompanied by the net release of 2-44 water molecules. The enhanced stabilities of CAP-DNA complexes with increased osmolality (decreased water activity) may contribute to the ability of E.coli cells to tolerate dehydration and/or high external salt concentrations.

The energy landscape of a fast-folding protein mapped by Ala-->Gly substitutions.

A moderately stable protein with typical folding kinetics unfolds and refolds many times during its cellular lifetime. In monomeric lambda repressor this process is extremely rapid, with an average folded state lifetime of only 30 milliseconds. A thermostable variant of this protein (G46A/G48A) unfolds with the wild-type rate, but it folds in approximately 20 microseconds making it the fastest-folding protein yet observed. The effects of alanine to glycine substitutions on the folding and unfolding rate constants of the G46A/G48A variant, measured by dynamic NMR spectroscopy, indicate that the transition state is an ensemble comprised of a disperse range of conformations. This structural diversity in the transition state is consistent with the idea that folding chains are directed towards the native state by a smooth funnel-like conformational energy landscape. The kinetic data for the folding of monomeric lambda repressor can be understood by merging the new energy landscape view of folding with traditional models. This hybrid model incorporates the conformational diversity of denatured and transition state ensembles, a transition state activation energy, and the importance of intrinsic helical stabilities.

Microsecond protein folding through a compact transition state.

Dynamic NMR methods have been employed to measure the folding and unfolding rate constants of two extremely fast-folding proteins. lambda 6-85, a truncated, monomeric form of the N-terminal domain of lambda repressor, refolds with a lifetime of approximately 250 microseconds. These methods have also been applied to a thermostable lambda 6-85 variant with alanine substituted for glycine residues 46 and 48 in the third helix (G46A/G48A). Both proteins exhibit linear ln (kf,u) versus [urea] plots, consistent with two-state folding for both proteins. When extrapolated to 0M urea, the data indicate that G46A/G48A folds with a lifetime of less than 20 microseconds. The slopes of the ln (kf,u) versus [urea] curves (mu and mf) indicate that the modest Gly-->Ala double mutation dramatically changes the transition state solvent accessibility. The transition state for lambda 6-85 has a fractional accessibility (mu/(mu-mf)) of 0.61, whereas the transition state for G46A/G48A is much more native-like, with a fractional accessibility of 0.16. The extraordinary change in the folding pathway that these mutations induce suggests that the intrinsic stability of helix 3 is an important determinant of the folding mechanism.

Bohr effects of the partially-ligated (CN-met) intermediates of hemoglobin as probed by quaternary assembly.

Free energies of quaternary assembly (dimers to tetramers) were determined for the 10 ligation species of CN-methemoglobin in the region of the alkaline Bohr effect (pH 7.0-9.5). Analysis of this database yielded the following principal findings: (1) At each pH, the nine CN-met species exhibit two distinct values of Bohr proton release and Bohr free energy. The two Bohr effects are found to distribute in a fashion that coincides with predictions of a symmetry rule (Ackers et al., 1992), i.e., the first value reflects a "tertiary Bohr effect" arising from ligation within the quaternary T tetramer and a second Bohr effect arises from the quaternary transition (T-->R) which occurs when both dimeric half-molecules acquire at least one ligated subunit. (2) The Bohr effects for CN-met ligation are in good agreement with previously-established Bohr effects for stepwise O2 binding under identical conditions (Chu et al., 1984). (3) In combination with recent studies which show that CN-met species [21] has a quaternary T structure (Daugherty et al., 1991; Doyle & Ackers, 1992; LiCata et al., 1993), the present results show that the "tertiary Bohr effect" within quaternary T exceeds the Bohr effect of dissociated dimers, as suggested by Lee and Karplus (1983). (4) The tertiary Bohr effect is found to account for the pH dependence of tertiary constraint energy, delta Gtc, which "pays" for ligand-binding cooperativity prior to the quaternary (T-->R) switchover. Possible origins of the tertiary Bohr effect and its relationship to the quaternary Bohr effect are considered.

Molecular code for cooperativity in hemoglobin.

Although tetrameric hemoglobin has been studied extensively as a prototype for understanding mechanisms of allosteric regulation, the functional and structural properties of its eight intermediate ligation forms have remained elusive. Recent experiments on the energetics of cooperativity of these intermediates, along with assignments of their quaternary structures, have revealed that the allosteric mechanism is controlled by a previously unrecognized symmetry feature: quaternary switching from form T to form R occurs whenever heme-site binding creates a tetramer with at least one ligated subunit on each dimeric half-molecule. This "symmetry rule" translates the configurational isomers of heme-site ligation into six observed switchpoints of quaternary transition. Cooperativity arises from both "concerted" quaternary switching and "sequential" modulation of binding within each quaternary form, T and R. Binding affinity is regulated through a hierarchical code of tertiary-quaternary coupling that includes the classical allosteric models as limiting cases.

Identification of the intermediate allosteric species in human hemoglobin reveals a molecular code for cooperative switching.

The 10 ligation species of human cyanomethemoglobin were previously found to distribute into three discrete cooperative free energy levels according to a combinatorial code (i.e., dependent on both the number and configuration of ligated subunits). Analysis of this distribution showed that the hemoglobin tetramer occupies a third allosteric state in addition to those of the unligated (T) and fully ligated (R) species. To determine the nature of the intermediate allosteric state, we have studied the effects of pH, temperature, and single-site mutations on its free energy of quaternary assembly, in parallel with corresponding data on the deoxy (T) and fully ligated (R) species. Results indicate that the intermediate allosteric tetramer has the deoxy (T) quaternary structure. This finding, together with the resolved energetic distribution of the 10 microstates reveals a symmetry rule for quaternary switching--i.e., switching from T to R occurs whenever a binding step creates a tetramer with one or more ligated subunits on each side of the alpha 1 beta 2 intersubunit contact. These studies also reveal significant cooperativity within each alpha 1 beta 1 dimer of the T-state tetramer. The ligand-induced tertiary free energy alters binding affinity within the T structure by 170-fold prior to quaternary switching.