Effect of colesevelam on faecal bile acids and bowel functions in diarrhoea-predominant irritable bowel syndrome.

BACKGROUND: About one-third of patients with IBS-diarrhoea (irritable bowel syndrome-D) have evidence of increased bile acid synthesis or excretion. AIMS: To assess effects of the bile acid sequestrant, colesevelam, on faecal excretion of BAs, hepatic BA synthesis and diarrhoea in IBS-D; to appraise whether individual or random stool samples accurately reflect 48-h total faecal bile acid excretion and proportions of the main bile acids excreted and to study the faecal fat excretion in response to colesevelam. METHODS: A single-centre, unblinded, single-dose trial of effects of colesevelam, 1875 mg [3 tablets (625 mg tablets)] orally, twice daily, for 10 days on total 48-h faecal bile acid excretion and fasting serum C4 (7α-hydroxy-4-cholesten-3-one; surrogate of hepatic bile acid synthesis). Stool diaries documented bowel functions for 8 days prior and 8 days during colesevelam treatment. Stool 48-h samples and fasting serum were collected for faecal fat, faecal bile acid and serum C4. RESULTS: Colesevelam was associated with significantly increased faecal total bile acid excretion and deoxycholic acid excretion, increased serum C4 and more solid stool consistency. There was a significant inverse correlation between number of bowel movements per week and the total bile acid sequestered into stool during the last 48 h of treatment. Random stool samples did not accurately reflect 48-h total or individual faecal bile acid excretion. Sequestration of bile acids by colesevelam did not increase faecal fat. CONCLUSIONS: Colesevelam increases delivery of bile acids to stool while improving stool consistency, and increases hepatic bile acid synthesis, avoiding steatorrhoea in patients with IBS-D. Overall effects are consistent with luminal bile acid sequestration by colesevelam.

Role of angiography in the detection of aortic branch vessel injury after blunt thoracic trauma.

PURPOSE: The occurrence of aortic arch branch vessel injury as an isolated occurrence or in association with aortic injury after blunt chest trauma has not been emphasized in the literature. The imaging evaluation is also controversial. METHODS: We reviewed thoracic aortograms of 166 patients examined at our institution from May 1995 to May 1999 performed after blunt thoracic trauma. We evaluated the aortograms for aortic and arch branch vessel injuries. Twenty-four injuries were detected and all patients had either a wide mediastinum demonstrated on plain radiographs (22 patients) or mechanism of injury conducive to aortic injury. RESULTS: Of the 166 patients, 24 (14%; 16 men, 8 women; mean age, 50 years) had aortic or arch branch vessel injuries. Isolated aortic injury occurred in 15 (9%) of 166 patients. Branch vessel injury occurred in 9 (5%) of 166 patients; seven patients (10 branch vessels) had isolated branch vessel injury and two patients (three branch vessels) had branch vessel injury associated with aortic injury. The injured branch vessels were brachiocephalic artery (four), left common carotid artery (four), left subclavian artery (three), right internal mammary artery (one), and left vertebral artery (one). The types of branch vessel injuries included intimal tears (nine vessels; 69%), and transection causing a pseudoaneurysm (four vessels; 31%). Revised Trauma Scores in patients with branch vessel injuries were 12 in seven patients and 11 and 4 in one each. CONCLUSION: We emphasize the angiographic findings in these patients that can at times be quite subtle. Awareness of the incidence of such injuries either in isolation or associated with aortic injury has implications regarding evaluation of this patient population with less invasive techniques such as CT or transesophageal echocardiography.

There is communication between all four Ca(2+)-bindings sites of calcineurin B.

We have used site-directed mutagenesis, flow dialysis, and Fourier transform infrared (FTIR) spectroscopy to study Ca(2+)-binding to the regulatory component of calcineurin. Single Glu-Gln(E --> Q) mutations were used to inactivate each of the four Ca(2+)-binding sites of CnB in turn, generating mutants Q1, Q2, Q3, and Q4, with the number indicating which Ca(2+) site is inactivated. The binding data derived from flow dialysis reveal two pairs of sites in the wild-type protein, one pair with very high affinity and the other with lower affinity Ca(2+)-binding sites. Also, only three sites are titratable in the wild-type protein because one site cannot be decalcified. Mutation of site 2 leaves the protein with only two titratable sites, while mutation of sites 1, 3, or 4 leave three titratable sites that are mostly filled with 3 Ca(2+) equiv added. The binding data further show that each of the single-site mutations Q2, Q3, and Q4 affects the affinities of at least one of the remaining sites. Mutation in either of sites 3 or 4 results in a protein with no high-affinity sites, indicating communication between the two high-affinity sites, most likely sites 3 and 4. Mutation in site 2 decreases the affinity of all three remaining sites, though still leaving two relatively high-affinity sites. The FTIR data support the conclusions from the binding data with respect to the number of titratable sites as well as the impact of each mutation on the affinities of the remaining sites. We conclude therefore that there is communication between all four Ca(2+)-binding sites. In addition, the Ca(2+) induced changes in the FTIR spectra for the wild-type and Q4 mutant are most similar, suggesting that the same three Ca(2+)-binding sites are being titrated, i.e., site 4 is the very high-affinity site under the conditions of the FTIR experiments.

Mutant protein in Huntington disease is resistant to proteolysis in affected brain.

The cause of Huntington disease pathophysiology is unknown, but a major hypothesis suggests that toxicity arises from the cleavage and accumulation of amino-terminal fragments containing an expanded polyglutamine region. In evaluating huntingtin protein (HD) from human brain, transgenic animals and cells, we observed, unexpectedly, that mutant HD is more resistant to proteolysis than normal HD. The N-terminal cleavage fragments we observed arise from the processing of normal HD and are sequestered by full-length mutant HD. Our results support a model in which inhibition of proteolysis of mutant HD leads to aggregation and toxicity through the sequestering of important targets, including normal HD.

Structures of apomyoglobin's various acid-destabilized forms.

The structures and the cold and hot melting thermodynamics of the acid- and salt-destabilized states of horse heart apomyoglobin (apoMb), including the E (extended) and various I forms, are studied using probes of tertiary structure (tryptophan fluorescence and FTIR spectroscopy) and secondary structure (far-UV CD and FTIR spectroscopy). These forms likely resemble early structures in the folding of the largely helical protein. Both the I and E forms retain the AGH core whereby the two ends of the protein are tied together with sufficient numbers of tertiary contacts, involving a number of hydrophobic residues, to show cooperative melting. The melting thermodynamics of E and I are distinctly different. E contains no other tertiary structure and probably little other secondary structure apart from the core. The more destabilized E form appears to contain "random" buried runs of polypeptide backbone which convert to alpha-helix in the I form(s). Most interestingly, E consists not of a single structure but is composed of a heterogeneous mixture of conformations, all showing corelike cooperative melting characteristics, and consisting presumably of varying contacts between the A portion of apomyoglobin and the G-H hairpin. These results bear on the energy landscape and structural features of the early part of apomyoglobin's folding pathway.

Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape.

An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.

A photolysis-triggered heme ligand switch in H93G myoglobin.

Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in < 5 micros. The results demonstrate that the H93G myoglobin cavity mutant has potential utility as a model system for studying the conformational energetics of ligand switching in heme proteins such as those observed in nitrite reductase, guanylyl cyclase, and possibly cytochrome c oxidase.

Human flap endonuclease-1: conformational change upon binding to the flap DNA substrate and location of the Mg2+ binding site.

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is a key enzyme in DNA replication and repair. FEN-1 recognizes the 5'-flap DNA structure and cleaves it, a specialized endonuclease function essential for the processing of Okazaki fragments during DNA replication and for the repair of 5'-end single-stranded tails from nicked double-stranded DNA substrates. Magnesium is a cofactor required for nuclease activity. We have used Fourier transform infrared (FTIR) spectroscopy to better understand how Mg2+ and flap DNA interact with human FEN-1. FTIR spectroscopy provides three fundamentally new insights into the structural changes induced by the interaction of FEN-1 with substrate DNA and Mg2+. First, FTIR difference spectra in the amide I vibrational band (1600-1700 cm(-1)) reveal a change in the secondary structure of FEN-1 induced by substrate DNA binding. Quantitative analysis of the FTIR spectra indicates a 4% increase in helicity upon DNA binding or about 14 residues converted from disordered to helical conformations. The observation that the residues are disordered without DNA strongly implicates the flexible loop region. The conversion to helix also suggests a mechanism for locking the flexible loop region around the bound DNA. This is the first direct experimental evidence for a binding mechanism that involves a secondary structural change of the protein. Second, in contrast with DNA binding, no change is observed in the secondary structure of FEN-1 upon Mg2+ binding to the wild type or to the noncleaving D181A mutant. Third, the FTIR results provide direct evidence (via the carboxylate ligand band at 1535 cm(-1)) that not only is D181 a ligand to Mg2+ in the human enzyme but Mg2+ binding does not occur in the D181A mutant which lacks this ligand.

CT diagnosis of acute flank pain from urolithiasis.

The use of noncontrast helical CT (NHCT) to assess patients with acute flank pain and hematuria for potential urinary tract stone disease was first reported in 1995. After several years of experience with the technique, sensitivity and specificity of NHCT has proven to be better than intravenous urography for evaluating ureteral stones. NHCT imaging findings for urinary calculi and the differential diagnosis are discussed in this article. Various extraurinary diseases found while using NHCT in searching for stone disease are addressed and illustrated. As experience with the use of NHCT has increased, clinicians have broadened the indications for this technique, which has a lower charge than standard CT, beyond the specific evaluation of urinary colic. This indication creep has increased the number of NHCT examinations ordered. It has also reduced the rate of stone positivity and increased the diagnostic yield for extraurinary disease.

Protein folding and unfolding on a complex energy landscape.

Recent theories of protein folding suggest that individual proteins within a large ensemble may follow different routes in conformation space from the unfolded state toward the native state and vice versa. Herein, we introduce a new type of kinetics experiment that shows how different unfolding pathways can be selected by varying the initial reaction conditions. The relaxation kinetics of the major cold shock protein of Escherichia coli (CspA) in response to a laser-induced temperature jump are exponential for small temperature jumps, indicative of folding through a two-state mechanism. However, for larger jumps, the kinetics become strongly nonexponential, implying the existence of multiple unfolding pathways. We provide evidence that both unfolding across an energy barrier and diffusive downhill unfolding can occur simultaneously in the same ensemble and provide the experimental requirements for these to be observed.

Trends in the use of unenhanced helical CT for acute urinary colic.

OBJECTIVE: Unenhanced helical CT for urolithiasis detection is a limited CT examination that was designed specifically for the detection of urolithiasis. The purpose of this study was to repeat a prior study to assess whether clinicians had broadened the indications and changed the yield and findings of unenhanced helical CT. MATERIALS AND METHODS: One hundred consecutive patients with suspected renal colic or flank pain referred for unenhanced helical CT were selected for this study. We reviewed the original radiographic reports for each patient and recorded the presence of ureteral calculi. Other urinary abnormalities and extraurinary lesions were also recorded and compared with the results of the previous study. RESULTS: In this study, 56% of the patients who underwent unenhanced helical CT had symptoms of urinary colic, and 44% of patients had unspecified flank pain, compared with 100% of patients with symptoms of urinary colic 1 year earlier. The sensitivity and specificity of unenhanced helical CT in detecting ureteral calculi were 96% and 99%, respectively. Ureteral calculi were identified in only 28% of the patients versus 49% of patients (p < .01) 1 year earlier. Extraurinary lesions were identified in 45% of the patients versus 16% (p < .01) 1 year before. CONCLUSION: As clinicians developed familiarity with this technique, the indications for performance of unenhanced helical CT were expanded with a consequent reduction in the rate of detection of stone disease and identification of an increased number of extraurinary lesions, which suggests a demand for emergency abdominal CT studies.

Abnormal calcification on plain radiographs of the abdomen.

The purpose of this pictorial review is to facilitate recognition and understanding of calcifications seen on conventional radiographs of the abdomen. Calcifications can be categorized by organ system and location in the abdomen. Both common and rare calcifications in the urinary tract, liver, gallbladder, spleen, pancreas, adrenal glands, digestive tract, genital tract, peritoneal cavity, and retroperitoneum are illustrated. Abnormal calcifications in the urinary tract are subcategorized by kidneys, ureters, bladder, and urethra. The density, shape, size, margins, pattern, position, and mobility of calcifications are emphasized for differential diagnoses.

Do's and don't's of percutaneous nephrostomy.

Percutaneous nephrostomy procedures generally are safe. The associated mortality rate is approximately 0.04%, and the incidence of important complications is 5% (2-4). To minimize complications, certain precautions always should be followed. First, radiologists should perform a preprocedural evaluation of the patient, with correction of marked coagulopathy or thrombocytopenia before all but the most emergent procedures. Second, antibiotics should be administered routinely before nephrostomy drainage; the choice of antibiotics can be based on the specific patient's risk factors for bacteriuria. To minimize the risk of clinically important renal vascular damage, radiologists should do the following: 1. Always achieve adequate visualization of the calices. 2. Identify a posterior calix for puncture that will give access to the appropriate segment of the kidney for anticipated procedures and allow safe creation of a tract. 3. Puncture below the 11th rib (and preferably below the 12th rib when feasible). 4. Puncture the tip of a posterior calix from a 20 degrees-30 degrees, posterolateral oblique approach to avoid major blood vessels. 5. Make a single-wall puncture of the calix. 6. Perform exchange transfusion for opacification of the renal pelvis and calices during percutaneous nephrostomy procedures to minimize the risk of sepsis. Overdistention can increase the likelihood of sepsis or retroperitoneal contamination. 7. Inject contrast material via a catheter placed over a wire to confirm the intracollecting system location of the entry. 8. Avoid unnecessary (complicated, prolonged) procedures in an infected, obstructed system. 9. Use only self-retaining drainage catheters to minimize the risk of inadvertent catheter dislodgment. 10. Create large-bore tracts with a balloon dilation system. By contrast, radiologists should not do the following: 1. Puncture above the 11th rib (unless all other avenues of approach have been exhausted). 2. Lose access to an obstructed kidney once the kidney has been punctured. Placement of a "safety" wire for all complex manipulations is recommended. 3. Panic if excessive bleeding or evidence of adjacent organ injury is seen. Excessive bleeding usually can be stopped with tract tamponade by using a balloon catheter advanced through the tract or with placement of an appropriate-sized nephrostomy tube to occlude the tract. If active bleeding continues or recurs, arteriography should be considered. The quantity of bleeding can be monitored with sequential hematocrit measurements. Almost all renal artery injuries can be treated with minimally invasive procedures, such as selective embolization of the branch artery involved, and this will lead to infarction of only a small segment of kidney, with preservation of functioning renal parenchyma. Injury to an adjacent organ usually can be treated nonsurgically (21,23). The most commonly injured extrarenal abdominal organ is the colon (Fig 6). On occasion, a percutaneous nephrostomy needle may traverse the retroperitoneal segment of the colon, and this type of injury generally can be treated nonsurgically, as well (23). If the colon has been traversed, adequate urinary drainage should be ensured before the transcolonic nephrostomy catheter is removed (so that a nephrocolonic fistula is not maintained). This can be done by placing a ureteral stent and a bladder catheter (18). Once adequate urinary drainage is provided, the nephrostomy catheter can be withdrawn into the colon and used as a percutaneous colostomy drain. The percutaneous colostomy tract should be allowed to mature for several days before this catheter is removed. In addition, appropriate antibiotics should be administered from the time a transcolonic tract is identified until the percutaneous tract has healed completely. Transthoracic entry can cause pneumothorax and pleural effusions. These should be treated only if they are large or cause symptoms (21). (ABSTRACT TRUNCATED)

Abnormal calcifications in the urinary tract.

A wide variety of calcifications may develop in the urinary tract. Calculi, the most common form of urinary tract calcification, are usually radiopaque due to their calcium content, whereas cystine stones tend to be less opaque. In cortical nephrocalcinosis, calcification may be spotty or may appear as a thin rim outlining the cortex. Intracystic calcification is usually thin and peripheral and is often described as having an "eggshell" appearance. In renal masses, pure central calcification usually indicates malignancy, although malignancy may also be present with pure peripheral calcification. An incomplete ring of calcification seen over the central portion of the kidney should suggest the presence of an abnormal vascular structure. A sloughed papilla may lead to calcification that is usually triangular or ring-shaped or has a broken rim pattern. Ureteral calculi usually have a uniform radiopacity, whereas phleboliths are often less opaque centrally. Like renal calculi, bladder calculi usually contain a calcium component; they may be laminated, faceted, spiculated, or seedlike in appearance. Urachal carcinoma is commonly associated with tumor calcification, which typically occurs at the dome of the bladder. Schistosomiasis of the bladder may produce mural calcification with a typical thin arcuate pattern and may be associated with calcification in other portions of the urinary tract. Although urinary tract calcifications may be difficult to characterize specifically, they can be classified according to location, appearance, and relation to various pathologic conditions.

GAA instability in Friedreich's Ataxia shares a common, DNA-directed and intraallelic mechanism with other trinucleotide diseases.

We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by improper DNA structure at the repeat region. Unlike CAG or CGG repeats, which form hairpins, GAA repeats form a YRY triple helix containing non-Watson-Crick pairs. As with hairpins, triplex mediates intergenerational instability in 96% of transmissions. In families with Friedreich's Ataxia, the only recessive trinucleotide disease, GAA instability is not a function of the number of long alleles, ruling out homologous recombination or gene conversion as a major mechanism. The similarity of mutation pattern among triple repeat-related diseases indicates that all trinucleotide instability occurs by a common, intraallelic mechanism that depends on DNA structure. Secondary structure mediates instability by creating strong polymerase pause sites at or within the repeats, facilitating slippage or sister chromatid exchange.

The core of apomyoglobin E-form folds at the diffusion limit.

The E-form of apomyoglobin has been characterized using infrared and fluorescence spectroscopies, revealing a compact core with native like contacts, most probably consisting of 15-20 residues of the A, G and H helices of apomyoglobin. Fast temperature-jump, time-resolved infrared measurements reveal that the core is formed within 96 micros at 46 degrees C, close to the diffusion limit for loop formation. Remarkably, the folding pathway of the E-form is such that the formation of a limited number of native-like contacts is not rate limiting, or that the contacts form on the same time scale expected for diffusion controlled loop formation.