Perforation of gastrointestinal tract by poorly conspicuous ingested foreign bodies: radiological diagnosis.

Perforation of gastrointestinal (GI) tract by ingested bone fragments, toothpicks and dentures is rare but remains an important life-threatening condition, and the outcomes are poorer when the diagnosis is delayed. Invariably, clinical and radiographic diagnosis is difficult as most patients will have no recollection of ingesting a foreign body, whereas these subtle objects are often not visible on radiographs. In search for the diagnosis, CT is the modality of choice, but ultrasound imaging may be first requested in patients presenting with symptoms of acute appendicitis, cholecystitis, pyelonephritis or pelvic inflammatory disease when an ingested foreign body is not considered. Although ultrasound has limited value in depicting a foreign body, it can frequently uncover secondary signs of perforation. However, the rarity of this condition combined with non-specific clinical presentation and the propensity of these small perforating objects to be subtle makes establishing the correct diagnosis by the radiologist challenging. Therefore, understanding of the appearances of GI perforation seen on CT images or general abdominal ultrasound will aid the radiologist in the diagnosis of this important yet often unsuspected condition. This will lead to earlier diagnosis and surgical management. In this article, we illustrate the spectrum of CT, radiographic and ultrasound imaging features seen in GI perforation caused by swallowed bone fragments, toothpicks, cocktail sticks and dentures.

Ultrasound detection of colonic polyps: perspective.

Colorectal cancer is often preventable if the precursor adenoma is detected and removed. Although ultrasound is clearly not one of the widely accepted screening techniques, this non-invasive and radiation-free modality is also capable of detecting colonic polyps, both benign and malignant. Such colon lesions may be encountered when not expected, usually during general abdominal sonography. The discovery of large colonic polyps is important and can potentially help reduce the incidence of a common cancer, whereas detection of a malignant polyp at an early stage may result in a curative intervention. This pictorial review highlights our experience of sonographic detection of colonic polyps in 43 adult patients encountered at our institutions over a 2-year period. 4 out of 50 discovered polyps were found to be malignant lesions, 3 polyps were hyperplastic, 1 polyp was a hamartomatous polyp and the rest were benign adenomas. The smallest of the detected polyps was 1.3 cm in diameter, the largest one was 4.0 cm (mean 1.7 cm; median 1.6 cm). In each case, polyps were discovered during a routine abdominal or pelvic examination, particularly when scanning was supplemented by a brief focused sonographic inspection of the colon with a 6-10 MHz linear transducer. In this paper, we illustrate the key sonographic features of different types of commonly encountered colonic polyps in the hope of encouraging more observers to detect these lesions, which may be subtle.

Influence of ethacrynic acid on glutathione S-transferase pi transcript and protein half-lives in human colon cancer cells.

Ethacrynic acid (EA) is a plant phenolic acid that is both an inhibitor and an inducer of glutathione S-transferase (GST) activity. To determine contributory factors in the increased GST activity caused by EA treatment, human colon carcinoma HT29 cells were compared with a cloned EA-resistant population (HT6-8) maintained in medium containing 72 microM EA. Several factors are involved in the increased expression of GST pi in HT6-8. For example, nuclear run-on experiments showed an approximately 2-fold increase in the rate of transcription of GST pi. In addition, the half-life of GST pi transcript was increased from 4.1 (wild type, HT29, HT4-1) to 8.4 hr. The half-life of GST pi protein was 1-2 hr in HT4-1 cells versus 8-9 hr in HT6-8 cells. When either human ovarian carcinoma cells (SKOV3) or human prostatic carcinoma cells (DU145) were treated with EA, the half-life of the GST pi transcript was also increased. The transcript half-lives of another thiol-metabolism enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and a phase II detoxification enzyme, dihydrodiol dehydrogenase (DDH), were also increased in HT6-8, SKOV3 and DU145 cells treated with EA. However, the half-lives of transcripts from "housekeeping genes," such as glyceraldehyde 3-phosphate dehydrogenase (G3PDH), beta-actin and beta-tubulin, were not changed in these cell lines following EA. Apparently, a number of coordinated factors are involved in EA-enhanced expression of GST pi and other detoxification enzymes.

Inhibition of big ET-1-induced pressor response by an orally active dual inhibitor of endothelin-converting enzyme and neutral endopeptidase 24.11.

The pharmacologic properties of CGS 26393, a prodrug of the endothelin-converting enzyme/neutral endopeptidase 24.11 inhibitor CGS 26303, were examined in conscious Sprague-Dawley rats. After oral administration of CGS 26393 at 30 mg/kg, the free concentrations of CGS 26303 in plasma were calculated to be 1.7 +/- 0.3, 1.2 +/- 0.2, and 0.31 +/- 0.05 microM at 4, 8, and 24 h, respectively. CGS 26393 inhibited the pressor response produced by exogenous big ET-1 in a dose-dependent manner. A 70% inhibition of the pressor response was observed when the prodrug was administered at 30 mg/kg p.o. As predicted by its pharmacokinetics, the inhibitory activity of CGS 26393 persisted for up to 8 h. These findings demonstrate that CGS 26393 in an orally active, long-acting ECE inhibitor in vivo.

Time course of glutathione S-transferase elevation in Walker mammary carcinoma cells following chlorambucil exposure.

Resistance of Walker 256 rat mammary carcinoma cells to chlorambucil has been shown to be accompanied by a specific increase in the A2-2 subunit of glutathione S-transferase (GST) (Buller et al., Mol Pharmacol 31: 575-578, 1987). Analysis of the time course of GST activity following chlorambucil exposure revealed a 7.5- and 3-fold elevation on day 7 post-treatment in Walker-sensitive (WS) and Walker-resistant (WR) cells, respectively. Flow activated cell sorting (FACS) analyses using antibodies specific for rat liver cytosolic GST supported these results and demonstrated the heterogeneous response of WS cells to chlorambucil exposure. The range of GST levels in drug-treated cells was very broad as compared to that of untreated cells. Transcripts for each class of GST (alpha, mu and pi) were quantified for days 1-9 post-treatment from densitometric scans of RNA slot blots. Elevations in GST alpha RNA preceded increases in GST activity (day 7) in both WS and WR cells. Because fluctuations in GSTA1-1 transcripts were not observed, it was concluded that the increased expression of the alpha class must be attributed to increases in GSTA2-2 transcripts. Amplification of the GST genes in drug-treated cells was not present. These results support the role of GSTA2-2 in the detoxification of chlorambucil. The time course of the cellular response to chlorambucil suggests that the elevation of GSTA2-2 transcripts following alkylating agent exposure may represent only one component of a series of events which collectively confer protection and lead to the establishment of drug resistance.

Increased levels of glutathione S-transferase pi transcript as a mechanism of resistance to ethacrynic acid.

Subpopulations of HT 29 human colon carcinoma cells (HT/M and HT/S) were selected for resistance to the glutathione S-transferase (GST) inhibitor ethacrynic acid (EA). Both clones displayed a 2-fold resistance to the selection agent and required its constant presence for the maintenance of the resistant phenotype. Purification and characterization of GST isoforms showed similar profiles in the wild-type (WT) and EA-resistant clones, with microheterogeneous forms of the pi isoenzyme detected in each case. Metabolism of EA in vitro in the presence of GSH and the isolated GST from each cell line was characterized by a biphasic disappearance of the parent drug; the initial rate at which each of these enzymes metabolized EA was similar. These enzymes also displayed similar Km values for 1-chloro-2,4-dinitrobenzene. However, the amount of GST isolated per total cellular protein was 3.0-fold in HT/M and 1.6-fold in HT/S relative to WT in the continuous presence of EA. Under these conditions GST activity was increased by 2.3-fold in HT/M and 3.2-fold in HT/S as were GSH levels (2.7- and 4.1-fold for HT/M and HT/S respectively). When EA was removed, enzyme activity and GSH concentrations decreased to values similar to those of the WT. Slot-blot and Southern analyses of the DNA gave no evidence of GST-pi-gene amplification or rearrangement. However, RNA analyses by both slot-blot and Northern studies indicate a 2.5-3.5-fold elevation in the GST pi transcript in the EA-resistant population. Results from these studies indicate that: (1) maintenance of the EA-resistant phenotype requires constant presence of the agent; (2) the 2-fold resistance to EA can be quantitatively related to a 2-3-fold increase in GST activity and amount which appears to be the result of a 2.5-3.5-fold elevation in GST transcript; (3) EA, a Michael-reaction acceptor, can induce GST at the transcriptional level.

Evidence for a glycoconjugate form of glutathione S-transferase pI.

The anionic form of glutathione S-transferase from human (GST pi) and rat (GST Yp) sources has been shown to exist in multiple forms which have similar molecular weights but different isoelectric points (pIs). Treatment with endoglycosidase H caused the acidic forms of GST Yp to be converted to proteins with more basic pIs as compared to the untreated control mixtures, suggesting that an N-linked mannose moiety containing acidic residues had been removed. Inability to detect these carbohydrates by techniques requiring unsubstituted vicinal hydroxyls further suggested acidic substitutions on the sugar moiety. GST pi/Yp carbohydrate modifications were also identified by differential staining procedures. These data represent the first indication that glycosylation of GST can occur. Additionally, this may offer an explanation for the often seen microheterogeneity within a class of GST isozymes.

Crystal and molecular structure of a DNA duplex containing the carcinogenic lesion O6-methylguanine.

The crystal and molecular structure of the first DNA duplex containing the carcinogenic lesion O6MeG has been determined to a resolution of 1.9 A and refined to an R factor of 19%. (d[CGC-(O6Me)GCG])2 crystallizes in the left-handed Z DNA form and has crystal parameters and conformational features similar to those of the parent sequence [d(CG)3]2. The methyl groups on O6 of G4 and G10 have C5-C6-O6-O6Me torsion angles of 73 degrees and 56 degrees, respectively, and protrude onto the major groove surface. The base-pairing conformation for the methylated G.C base pairs is of the Watson-Crick type as opposed to a wobble-type conformation that had been proposed in a B DNA fragment. As in other Z DNA structures, a spine of hydration is seen in the minor groove.

Repair of oligodeoxynucleotides containing O6-methylguanine by O6-alkylguanine-DNA-alkyltransferase.

O6-Alkylguanine-DNA-alkyltransferase is a DNA repair protein known to carry out the transfer of alkyl groups from the O6-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. We have examined the ability of this protein isolated from either E. coli or mammalian cells to perform this repair reaction in short oligodeoxynucleotides. Dodecadeoxynucleotides of the sequence 5'-dCGNGAATTCm6GCG-3' where N is any one of the normal four bases were all repaired very rapidly by the protein with 50% repair in less than 15 s at 0 degree C. The hexadeoxynucleotide 5'-dCGCm6GCG-3' was repaired slightly more slowly with 50% removal taking 7 min at 0 degree C and 1.5 min at 37 degrees C. The tetradeoxynucleotide 5'-dTm6GCA-3' was also a substrate but was repaired much more slowly requiring 45 min for 50% repair at 37 degrees C. These results indicate that (a) the AGT has a strong but not absolute preference for double-stranded DNA substrates; (b) the repair of O6-methylguanine is independent of the base opposite the lesion; and (c) that oligodeoxynucleotides as short as tetramers are substrates for repair by this protein.

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes.

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.

Specifically alkylated DNA fragments. Synthesis and physical characterization of d[CGC(O6Me)GCG] and d[CGT(O6Me)GCG].

Two hexamer DNA fragments containing a carcinogenic modified base, O6-methyl guanine, have been synthesized by a solid-phase phosphotriester method, in which the unmodified guanine residues present were O6 protected with the 4-nitrophenylethyl group. These two alkylated oligonucleotides were found to have similar Tm's about 40 degrees lower than the unmodified parent compound, d(CG)3. Moreover, the presence of the (O6Me)G appears to inhibit the B leads to Z transition, as determined by CD spectroscopy.

Synthesis and physical characterization of the self-complementary, alternating pyrimidine/purine hexanucleotide d[CGTACG].

The hexanucleotide d(CGTACG) has been synthesized by a phosphotriester method in which, for the first time, an O-6 protected deoxyguanosine derivative has been used to avoid side reactions of the guanine residues. Conformational analysis by circular dichroism shows that d(CGTACG) maintains a B form under conditions (5 M NaCl) where the all C/G hexanucleotide d(CG)3 adopts a Z form, even though d(CGTACG) is a fully alternating pyrimidine/purine molecular. The delta H for the helix-to-coil transition has been measured.