DNA damage recognition and repair pathway coordination revealed by the structural biochemistry of DNA repair enzymes.

Cells have evolved distinct mechanisms for both preventing and removing mutagenic and lethal DNA damage. Structural and biochemical characterization of key enzymes that function in DNA repair pathways are illuminating the biological and chemical mechanisms that govern initial lesion detection, recognition, and excision repair of damaged DNA. These results are beginning to reveal a higher level of DNA repair coordination that ensures the faithful repair of damaged DNA. Enzyme-induced DNA distortions allow for the specific recognition of distinct extrahelical lesions, as well as tight binding to cleaved products, which has implications for the ordered transfer of unstable DNA repair intermediates between enzymes during base excision repair.

Prevalence of short segments of specialized columnar epithelium in distal esophagus: association with gastroesophageal reflux.

BACKGROUND AND AIMS: Recent studies have reported high prevalence rates of short segments of specialized columnar epithelium (SCE) in the distal esophagus. The association of SCE with gastroesophageal reflux disease is not well established. We studied the prevalence and associations of short segments of SCE in the distal esophagus amongst Indians. METHODS: 271 patients (mean age 36 [14] y; 160 men) undergoing diagnostic upper gastrointestinal endoscopy were interviewed regarding symptoms of gastroesophageal reflux, and history of medications, smoking or chewing tobacco and alcohol ingestion. At endoscopy, presence and grade of esophagitis and hiatus hernia were recorded. One biopsy each was taken from the squamocolumnar junction and 2 cm proximal to it. Biopsies were stained with hematoxylin/eosin and alcian blue/periodic acid-Schiff. The pathologist was blinded to the clinical and endoscopic data. RESULTS: Short segments of SCE in the distal esophagus were present in 16/271 (6%; CI 5.03-6.97) patients. Increasing age (p<0.01), and endoscopic (p<0.01) and histologic (p<0.001) esophagitis were associated with its presence, whereas symptoms of gastroesophageal reflux, smoking, tobacco chewing, use of alcohol or non-steroidal anti-inflammatory drugs, and hiatus hernia were not. One patient with SCE had dysplasia. CONCLUSION: Prevalence of short segments of SCE in the distal esophagus amongst Indians is low and is usually associated with inflammation in the esophagus.

Lessons learned from structural results on uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) functions as a sentry guarding against uracil in DNA. UDG initiates DNA base excision repair (BER) by hydrolyzing the uracil base from the deoxyribose. As one of the best studied DNA glycosylases, a coherent and complete functional mechanism is emerging that combines structural and biochemical results. This functional mechanism addresses the detection of uracil bases within a vast excess of normal DNA, the features of the enzyme that drive catalysis, and coordination of UDG with later steps of BER while preventing the release of toxic intermediates. Many of the solutions that UDG has evolved to overcome the challenges of policing the genome are shared by other DNA glycosylases and DNA repair enzymes, and thus appear to be general.

Uracil-DNA glycosylase-DNA substrate and product structures: conformational strain promotes catalytic efficiency by coupled stereoelectronic effects.

Enzymatic transformations of macromolecular substrates such as DNA repair enzyme/DNA transformations are commonly interpreted primarily by active-site functional-group chemistry that ignores their extensive interfaces. Yet human uracil-DNA glycosylase (UDG), an archetypical enzyme that initiates DNA base-excision repair, efficiently excises the damaged base uracil resulting from cytosine deamination even when active-site functional groups are deleted by mutagenesis. The 1.8-A resolution substrate analogue and 2.0-A resolution cleaved product cocrystal structures of UDG bound to double-stranded DNA suggest enzyme-DNA substrate-binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally stabilized closing of UDG enforces distortions of the uracil and deoxyribose in the flipped-out nucleotide substrate that are relieved by glycosylic bond cleavage in the product complex. This experimentally defined substrate stereochemistry implies the enzyme alters the orientation of three orthogonal electron orbitals to favor electron transpositions for glycosylic bond cleavage. By revealing the coupling of this anomeric effect to a delocalization of the glycosylic bond electrons into the uracil aromatic system, this structurally implicated mechanism resolves apparent paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite mutational removal of active-site functional groups. These UDG/DNA structures and their implied dissociative excision chemistry suggest biology favors a chemistry for base-excision repair initiation that optimizes pathway coordination by product binding to avoid the release of cytotoxic and mutagenic intermediates. Similar excision chemistry may apply to other biological reaction pathways requiring the coordination of complex multistep chemical transformations.

DNA repair mechanisms for the recognition and removal of damaged DNA bases.

Recent structural and biochemical studies have begun to illuminate how cells solve the problems of recognizing and removing damaged DNA bases. Bases damaged by environmental, chemical, or enzymatic mechanisms must be efficiently found within a large excess of undamaged DNA. Structural studies suggest that a rapid damage-scanning mechanism probes for both conformational deviations and local deformability of the DNA base stack. At susceptible lesions, enzyme-induced conformational changes lead to direct interactions with specific damaged bases. The diverse array of damaged DNA bases are processed through a two-stage pathway in which damage-specific enzymes recognize and remove the base lesion, creating a common abasic site intermediate that is processed by damage-general repair enzymes to restore the correct DNA sequence.

Gastric emptying of solids in long-term NSAID users: correlation with endoscopic findings and Helicobacter pylori status.

OBJECTIVE: Prostaglandins regulate gastric motor function. Inhibition of prostaglandins by nonsteroidal antiinflammatory drugs (NSAIDs) may alter gastric emptying. To study gastric emptying of solids and its relation to endoscopic findings and Helicobacter pylori in patients receiving long-term NSAIDs, we undertook this study. METHODS: Ninety-five patients with arthritis, 65 taking long-term NSAIDs (Group I) and 30 not taking NSAIDs (Group II) were studied. Presence of dyspeptic symptoms was determined using a questionnaire. Mucosal damage was determined by endoscopy. H. pylori was detected by antral biopsies for rapid urease test and histology. Gastric emptying for solids was evaluated using a scintigraphic method. Thirty healthy volunteers were used as controls for gastric emptying (Group III). Patients with peptic ulcer were excluded from the analysis of gastric emptying. Logistic regression analysis was performed to identify predictive factors for gastric emptying. RESULTS: Nineteen patients from Group I with peptic ulcers were excluded. Dyspeptic symptoms were seen in 24 (52%) Group I and seven (23%) Group II patients. Gastroduodenal erosions were seen in 10 (21.7%) Group I patients and four (13.3%) Group II patients. H. pylori was detected in 17 patients in Group I (36.9%) and Group II (56.6%). Gastric emptying was delayed in 24 (52%) Group I patients, six (20%) Group II patients (p < 0.001), and in none of the Group III controls. The mean gastric emptying times were 99.5 (15.6) min and 89 (17.7) min for Groups I and II, respectively (p < 0.05). Endoscopic damage was found with similar frequency in Group I patients with delayed or normal gastric emptying. H. pylori infection was present in 37.5% Group I patients with delayed gastric emptying and in 36.3% with normal gastric emptying (p = ns). Logistic regression analysis identified NSAID therapy as the single factor most predictive of delayed gastric emptying. CONCLUSION: Delayed gastric emptying was seen in 52% of patients on long-term NSAID therapy.

Envisioning the molecular choreography of DNA base excision repair.

Recent breakthroughs integrate individual DNA repair enzyme structures, biochemistry and biology to outline the structural cell biology of the DNA base excision repair pathways that are essential to genome integrity. Thus, we are starting to envision how the actions, movements, steps, partners and timing of DNA repair enzymes, which together define their molecular choreography, are elegantly controlled by both the nature of the DNA damage and the structural chemistry of the participating enzymes and the DNA double helix.

High frequency of bacteremia with endoscopic treatment of esophageal varices in advanced cirrhosis.

OBJECTIVES: The fregency of bacteremia after endoscopic variceal band ligation (EVL) is reported to be lower when compared to that after endoscopic variceal sclerotherapy (EVS). However, there are conflicting reports on the infectious sequelae after EVL. AIM: To compare the frequency on bacteremia and infectious sequelae after EVL and EVS in patients with cirrhosis of liver. METHODS: Bacteremia and infectious sequelae were studied in 32 sessions of EVL in 18 cirrhotic patients (Child-Pugh class A-6, B-5, C-7), 30 sessions of EVS in 22 cirrhotic patients (Child-Pugh class A-2, B-5, C-15) and 14 diagnostic upper gastrointestinal endoscopies. Blood cultures were collected before, during and 30 minutes after the procedure. Patients were observed for infectious sequelae during subsequent hospitalization. RESULTS: Before the procedure, bacteremia was present in 7/62 (11%) sessions. Significant bacteremia during and 30 min post-procedure developed in 8/32 (25%) and 12/30 (40%) of EVL and EVS sessions, respectively (p = ns), and in 1/14 (7%) upper gastrointestinal endoscopy sessions. There was more frequent bacteremia with severe liver disease (Child-Pugh class A-0/6, B-1/5. C-7/21; p = 0.09) in the EVL but not in the EVS group (Child-Pugh class A-1/3, B-2/5, C-9/22; p = ns). The incidence was higher with emergency sclerotherapy compared to elective sclerotherapy (6/8 [75%] vs 6/22 [27%]; p <0.01). One patient in the EVS group developed spontaneous bacterial peritonitis. CONCLUSIONS: Bacteremia occurs frequently following EVL and EVS in patients with advanced liver diseases. In the EVS group it is more common after emergency sclerotherapy. This bacteremia is rarely associated with significant infectious sequelae.

MutY catalytic core, mutant and bound adenine structures define specificity for DNA repair enzyme superfamily.

The DNA glycosylase MutY, which is a member of the Helix-hairpin-Helix (HhH) DNA glycosylase superfamily, excises adenine from mispairs with 8-oxoguanine and guanine. High-resolution crystal structures of the MutY catalytic core (cMutY), the complex with bound adenine, and designed mutants reveal the basis for adenine specificity and glycosyl bond cleavage chemistry. The two cMutY helical domains form a positively-charged groove with the adenine-specific pocket at their interface. The Watson-Crick hydrogen bond partners of the bound adenine are substituted by protein atoms, confirming a nucleotide flipping mechanism, and supporting a specific DNA binding orientation by MutY and structurally related DNA glycosylases.

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA.

Three high-resolution crystal structures of DNA complexes with wild-type and mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and damage-general BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endonuclease, which catalyzes the first damage-general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions.

Bilioma secondary to choledocholithiasis.

Bilioma secondary to choledocholithiasis is rare. We report a patient in whom a large common bile duct stone was responsible for leak from the infraduodenal segment of the bile duct. Choledochotomy with extraction of stone followed by T-tube drainage of the bile duct and evacuation of the bilioma resulted in complete recovery.

Impact of human immunodeficiency virus infection on abdominal tuberculosis in western India.

We studied the seroprevalence of human immunodeficiency virus infection in patients with pulmonary tuberculosis and abdominal tuberculosis. We also assessed the clinical characteristics, risk factors, tuberculin status, site, and response to therapy of abdominal tuberculosis in human immunodeficiency virus (HIV)-seropositive and HIV-seronegative patients. Volunteer blood donors (n = 8,395), patients with pulmonary tuberculosis (n = 387), and patients with abdominal tuberculosis (n = 108) were screened for HIV 1 and/or HIV 2 by enzyme-linked immunosorbent assay (ELISA; Torrent, India) and positivity reconfirmed by a repeat ELISA and Western blot test. The HIV seroprevalence in the abdominal tuberculosis patients (16.6%) was significantly higher compared with those with pulmonary tuberculosis (6.9%, p < 0.05) and volunteer blood donors (1.4%, p < 0.01). Absolute lymphocyte counts did not differ between the HIV-seropositive and HIV-seronegative patients (2,044.94 +/- 830 vs 2,261.34 +/- 805/mm3, p = NS). The Mantoux reaction was larger in the HIV-seronegative group as compared with the HIV-seropositive group (14.8 mm vs. 9.5 mm, p < 0.05). Tuberculosis patients responded well to conventional antituberculosis drugs in standard doses regardless of their HIV status.

PIP: Human immunodeficiency virus (HIV) infection dramatically increases the risk of progression of latent tuberculosis infection. A case-control study conducted during 1992-95 at an urban teaching hospital in Mumbai, India, investigated the seroprevalence of HIV infection in patients with pulmonary and abdominal tuberculosis. Enrolled were 387 patients with active pulmonary tuberculosis, 108 with abdominal tuberculosis, and 8359 volunteer blood donors. The HIV seroprevalence rates in these 3 groups were 6.9%, 16.6%, and 1.4%, respectively. The majority of HIV-infected abdominal tuberculosis patients were in the early clinical stages of the disease and not significantly immunosuppressed. These patients had higher rates of lymphatic and hepatic tuberculosis than seronegative patients, suggesting disease dissemination. All tuberculosis patients, regardless of HIV status, responded well to antituberculous drugs in standard doses. These findings suggest that all patients with pulmonary and abdominal tuberculosis should be screened for HIV. In addition, long-term chemoprophylaxis in HIV-infected persons from tuberculosis-endemic areas should be considered.

Base excision repair enzyme family portrait: integrating the structure and chemistry of an entire DNA repair pathway.

DNA base excision repair (BER) is essential to preserving the integrity of the genome. Recent crystallographic studies of representatives from each enzyme class required for BER reveal clues to the structural basis of an entire DNA repair pathway.

Long-term anti-inflammatory drug use and Helicobacter pylori infection: a clinical, endoscopic and histological study.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori are independent risk factors for gastroduodenal damage and peptic ulcer. OBJECTIVE: To study the frequency and effect of H pylori infection on gastroduodenal mucosa in patients on long-term NSAID use. METHODS: A total of 125 subjects were studied: 65 patients (Group 1) on NSAID therapy (> 6 months), 30 patients (Group 2) with arthritic disorders prior to starting NSAID therapy, and 30 healthy volunteers (Group 3). Dyspeptic symptoms were evaluated using a questionnaire. All patients underwent endoscopy and antral and duodenal biopsies were obtained to assess the extent of gastroduodenal damage and H pylori status. RESULTS: H pylori infection was less frequent in Group 1 (37%) compared to Group 2 (57%, p = ns) and 3 (60%, p < 0.05). Among Group 1 patients, H pylori infection did not increase the risk of gastroduodenal damage (52% vs 45%) or ulceration (32% vs 27%). Group 1 patients with H pylori infection were more likely to be symptomatic (48% vs 27%) and have chronic active gastritis (76% vs 12%) and chronic active duodenitis (68% vs 5%). Gastric metaplasia was seen only in patients with H pylori infection, chronic active gastritis and duodenitis. Chemical gastritis was observed more commonly in Group 1 (34% vs 3%) compared to Group 2; its was not seen in Group 3. H pylori infection was less commonly observed in patients with chemical gastritis (8% vs 50%). CONCLUSION: Patients on long-term NSAIDs are not at increased risk of H pylori infection. Presence H pylori infection is not associated with increased risk of gastroduodenal damage in these patients. H pylori infection correlated with presence of chronic active gastritis, and NSAID with presence of chemical gastritis.

Frequency analysis of large CAG/CTG trinucleotide repeats in schizophrenia and bipolar affective disorder.

Much interest has recently been focussed on the possibility of the involvement of unstable DNA in the etiology of schizophrenia and bipolar affective disorder (BPAD), following several publications that report increases in frequency of large CAG/CTG repeats in affected individuals. Using the Repeat Expansion Detection (RED) technique, we have performed a matched control pair analysis for both disorders. No significant differences in CAG/CTG repeat sizes were observed for 52 bipolar affecteds and matched controls (P = 0.15), and borderline significance was observed for 54 schizophrenia affecteds and matched controls (P = 0.05), using a (CTG)10 oligonucleotide (one-tailed t-tests for paired samples). Furthermore, using a (CTG)17 oligonucleotide, no significant differences were observed for 58 bipolar affecteds and 55 schizophrenia affecteds compared to 81 unmatched controls. No significant sex effect was observed for either group, and no significant differences in repeat size were found for responders and non-responders to drug treatments. More importantly, there was no significant correlation (either positive or negative) between age of onset of disease and size of repeat. We thus cannot conclude that CAG/CTG trinucleotides are involved in psychotic disorders and that either the differences observed in similar studies may be the result of population stratification, or that the increased frequency of larger repeats amongst affected individuals is a much smaller effect than previously thought.

Postoperative delirium in the elderly.

Postoperative delirium is common in the elderly in the postoperative period. It can result in increased morbidity, delayed functional recovery, and prolonged hospital stay. In surgical patients, factors such as age, alcohol abuse, low baseline cognition, severe metabolic derangement, hypoxia, hypotension, and type of surgery appear to contribute to postoperative delirium. Anesthetics, notably anticholinergic drugs and benzodiazepines, increase the risk for delirium. Despite the above recommendations, postoperative delirium in the elderly is poorly understood. Clearly, further studies are needed to determine the risk and long-term outcome of delirium in the elderly population. Research is also needed to define the effects of hypoxemia on cerebral function and whether oxygen therapy has any benefits. The geriatric-anesthesiologic intervention program of pre- and postoperative geriatric assessment, early surgery, thrombosis prophylaxis, oxygen therapy, prevention and treatment of perioperative decrease in blood pressure, and vigorous treatment of any postoperative complications showed some promise, but further definitive studies are needed.