In vivo footprinting of a muscle specific enhancer by ligation mediated PCR.

In vivo protein-DNA interactions at the developmentally regulated enhancer of the mouse muscle creatine kinase (MCK) gene were examined by a newly developed polymerase chain reaction (PCR) footprinting procedure. This ligation mediated, single-sided PCR technique permits the exponential amplification of an entire sequence ladder. Several footprints were detected in terminally differentiated muscle cells where the MCK gene is actively transcribed. None were observed in myogenic cells prior to differentiation or in nonmuscle cells. Two footprints appear to correspond to sites that can bind the myogenic regulator MyoD1 in vitro, whereas two others represent muscle specific use of apparently general factors. Because MyoD1 is synthesized by undifferentiated myoblasts, these data imply that additional regulatory mechanisms must restrict the interaction between this protein and its target site prior to differentiation.

Myt1: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15.

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phosphorylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1, a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggested that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control.

Genomic sequencing and methylation analysis by ligation mediated PCR.

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.

Cell cycle regulation of a Xenopus Wee1-like kinase.

Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase.

Short-segment intestinal interposition of the distal esophagus.

Esophageal replacement remains a challenge. Colon and jejunum provide alternative conduits to replace the lower esophagus when stomach is not suitable. Between 1971 and 1991, 41 patients underwent short-segment interposition of the esophagus with jejunum or colon. Indications were failed antireflux procedures (n = 21), nondilatable stricture (n = 9), achalasia (n = 2), moniliasis (n = 2), Barrett's esophagus with carcinoma in situ (n = 2), hemorrhagic esophagitis after esophagogastrectomy (n = 1), motility disorder (n = 1), instrumental perforation (n = 1), carcinoma (n = 1), and leiomyosarcoma (n = 1). Thirty-one patients (75.6%) had prior surgical procedures. Interposition with colon was performed in 22 patients and with jejunum in 19. Major complications occurred in 45% after colon interposition (10/22) and hospital mortality was 4.5% (1/22). Major complications after jejunal interposition occurred in 31% (6/19) and hospital mortality was 10.5% (2/19). A contained anastomotic leak occurred in 1 patient, perforation of a colon segment in 1, and jejunal graft necrosis in a third. Late functional results in 34 patients with a mean follow-up of 87 months were excellent or good in 26, fair in 5, and poor in 1. Colon interposition failed to improve symptoms in 2 patients with gastrointestinal motility disorders. Six patients underwent manometry and barium food provocation study. Two colon segments and 3 jejunal interpositions were hypoperistaltic or aperistaltic according to manometry. There was 1 case of aperistaltic jejunum with a distended afferent loop. When stomach is not available, successful palliation of swallowing can be accomplished with either jejunum or colon. Surgeons involved in the management of esophageal disease should be familiar with the technical details of both procedures.

Dissolution of calcified gallstones. Part I. Correlation of in vitro dissolution kinetics in methyl tert-butyl ether with patterns of calcification by computed tomography.

RATIONALE AND OBJECTIVES: The authors evaluated the relationship between stone computed tomography (CT) attenuation patterns and the kinetics of dissolution with methyl tertbutyl ether (MTBE). METHODS: Single moderately and heavily calcified gallstones from 40 patients were selected from a gallstone library and classified for pattern of calcification by in vitro CT scan (dense, rim, core, and laminated). Each stone was placed in a 10-mL aliquot of MTBE for 24 hours. Stone residue was blotted dry and weighted at 8, 16, and 24 hours. Results were normalized with respect to stone size. RESULTS: Only 1 of 40 (4%) specimens dissolved to particulate matter that was smaller than 2 mm. All (6 of 6) stones that were densely calcified showed virtually no dissolution. The rate of gallstone dissolution varied temporally within the rim, core, and laminated stone categories and was related to the composition of the layer exposed to the solvent at any given time. CONCLUSION: The success and rate of dissolution may be predicted by the pattern of calcification as determined by computed tomography (CT).

Dissolution of calcified gallstones. Part II. Evaluation of edetic acid preparations for dissolution of residue after in vitro methyl tert-butyl ether treatment.

RATIONALE AND OBJECTIVES: The authors assessed the potential of edetic acid (EDTA) preparations to dissolve the residue of calcified gallstones partially treated with methyl tert-butyl ether (MTBE). METHODS: Nineteen triplets (57 gallstones) were submitted to dissolution in EDTA, urea-EDTA, and an MTBE control for 48 hours after initial partial dissolution in MTBE for 24 hours. Results were compared with findings at specimen computed tomography and crystallographic analysis. All data were corrected for differences in stone size. RESULTS: In all three treatment groups (EDTA, urea-EDTA, MTBE), almost identical dissolution outcomes were observed within each triplet. Most triplets that dissolved displayed a laminated or a core-calcification pattern and consisted primarily of cholesterol. Specimens that dissolved poorly in all three groups displayed dense calcifications or thick calcified rims and were classified as pigment stones. CONCLUSION: Because no statistically significant differences in dissolution were found among the EDTA, urea-EDTA, and MTBE treatments, we conclude that EDTA preparations are not superior to the continued use of MTBE for dissolution of residue after initial MTBE treatment.

Small chest-tube drainage followed by bleomycin sclerosis for malignant pleural effusions.

OBJECTIVE: To review our experience with bleomycin sclerotherapy as treatment of pleural effusions due to gynecologic malignancies. METHODS: Twenty-one women with histologically documented malignant effusions (16 ovarian carcinoma, three uterine sarcoma, and two cervical cancer) were treated with small flexible chest-tube drainage followed by intrapleural bleomycin sclerotherapy (60 units). RESULTS: After placement of the chest tube, suction was required for a median of 5 days (range 3-12) before output was low enough (less than 100 mL/24 hours) to instill bleomycin. Among 24 treated effusions, there was a 71% overall response rate, including ten complete responses (42%) and seven partial responses (29%); seven effusions (29%) did not respond to therapy. Six of the seven patients whose effusions did not respond to bleomycin died of disease within 2 months of attempted sclerotherapy. Fever was the most common side effect, occurring in 13 of 21 patients (62%) following instillation of bleomycin. Pain during sclerosis was reported by only two patients. CONCLUSIONS: Bleomycin sclerotherapy after small flexible chest-tube drainage of malignant pleural effusions is an effective technique, with minimal adverse reactions, for controlling effusions that develop in women with gynecologic malignancies.

Percutaneous drainage of diverticular abscess. An adjunct to surgical therapy.

We undertook this study to determine whether a computed tomography-guided, percutaneous preoperative drainage of a peridiverticular abscess can safely allow a one-stage procedure in patients requiring surgery for acute diverticulitis. In 17 patients evaluated prospectively by computed tomography, thin-needle aspiration demonstrated purulent fluid collection in 11 patients. Percutaneous catheter drainage was undertaken in eight patients. In the three remaining patients, the abscess was either too small to warrant drainage or no safe access route was present. Seven of eight patients had a single-stage resection within one to three weeks of percutaneous catheter drainage. There were no complications. Our studies suggest that a combined radiological-surgical approach has the potential to reduce morbidity and hospital costs without increasing mortality in the management of perforated colon diverticulitis with associated abscess formation.

Eclectic drainage of subphrenic abscesses.

In 196 cases of subphrenic abscess from 1964 through 1979, 56% were attributable to gastric, hepatic, and colonic disease or surgery. Posttraumatic abscesses in younger patients became more frequent. Synchronous suprahepatic and subhepatic abscesses or bilateral abscesses accounted for 19%. Streptococci, Escherichia coli, Klebsiella, and Bacteroides species were the most frequently isolated organisms. Although the overall mortality rate was 40%, the surgical mortality rate decreased from 33% initially to 17% recently. The mortality rate of transperitoneal drainage decreased from 41% to 16%. From 1980 through early 1983, a success rate of 84%, with no fatalities, was achieved in percutaneous radiologic drainage of 25 unilocular abscesses. At present, radiologically guided drainage should be considered for unilocular abscesses and some bilocular ones. Although extraperitoneal, extrapleural surgical drainage remains an expeditious form of treatment, it may give way to radiologic drainage. Transperitoneal drainage is preferable for multifocal abscesses and for many abscesses secondary to complications of intraabdominal surgery.

Abdominal abscess. Imaging and intervention.

The diagnosis and management of patients with abdominal abscesses continue to challenge those involved in imaging and interventional techniques. In this article, the authors discuss the use of ultrasonography and CT scans in the work-up of patients suspected of having abdominal abscesses and describe techniques useful for their management preoperatively.

Pleural interventions. Indications, techniques, and clinical applications.

Pleural interventions represent one of the newest frontiers of interventional radiology. Many of the data are preliminary, but it is tempting to postulate that future side-by-side comparisons would prove these innovations superior to unguided techniques. Each procedure utilizes similar principles of pleural intervention yet emphasizes unique issues pertinent to the disease processes involved.

Thyroid and parathyroid ultrasonography.

The introduction of high-resolution real-time small-parts sonography has made imaging the superficial structures of the neck a practical procedure. This article reviews the normal anatomy of the neck as well as the benign and malignant diseases affecting the thyroid and parathyroid glands.

Percutaneous balloon dilatation of benign biliary strictures.

Percutaneous biliary dilatation is an effective alternative to surgical management of benign biliary strictures that has low morbidity and no reported mortality. Reported success rates for this procedure range from 40% to 90% depending on the size of the series, the type of patient being treated, and the length of follow-up period. The procedure is done in the fluoroscopy suite with an angioplasty balloon catheter. Transhepatic access is most common, but the procedure may be done via existing T-tube tracts or specially created jejunal loops. As the frequency of radical liver surgery such as liver transplant and radical trisegmentectomy rises, so too, the rate of biliary stricture is likely to rise, making percutaneous balloon dilatation an increasingly important tool in the interventional radiologist's armamentarium.

The uses of diagnostic ultrasound in the thorax.

Modern real-time gray scale ultrasonography has contributed new diagnostic and therapeutic information in patients with diseases of the lungs and pleura. This article will discuss the ways in which ultrasound is most clinically useful in patients with thoracic disease.

Early surgical débridement of symptomatic pancreatic necrosis is beneficial irrespective of infection.

In order to assess the recent trend of nonoperative management of pancreatic necrosis, we reviewed 82 variables in 73 consecutive patients with symptomatic necrotizing pancreatitis. The mortality rate for the series was 25% (18 of 73). The only preintervention variables that correlated with mortality were APACHE II score greater than 15 (p = 0.01), preintervention blood transfusion (p less than 0.001), respiratory failure (p less than 0.001), and shock (p less than 0.01). Patients who developed recurrent sepsis following the initial intervention had a significantly higher mortality rate (17 of 34) than those who did not (1 of 39) (p less than 0.001). The rate of recurrent sepsis varied widely among individual surgeons and correlated with APACHE II score. The presence of infected versus noninfected necrosis did not correlate significantly with outcome. When percutaneous radiologically guided drainage was the initial therapeutic modality (n = 6), recurrent sepsis requiring surgical drainage inevitably occurred. Patients treated with percutaneous drainage (often in combination with surgical drainage) had a longer hospital stay (82 versus 42 days, p less than 0.001), spent more days in the intensive care unit (31 versus 6 days, p less than 0.001), and required more days of total parenteral nutrition (57 versus 27 days, p less than 0.001) than those treated solely by surgical means. We conclude that aggressive initial surgical débridement should be the first step in managing symptomatic pancreatic necrosis and that the presence of infection should not be the sole determinant of intervention.

Extracorporeal shock-wave lithotripsy of gallstones: Boston experience with the Dornier MPL 9000 lithotripter.

After the success of shock-wave therapy for fragmentation of renal stones, machines for shock-wave therapy of gallbladder stones were quick to follow. In Munich, gallstones were successfully treated in 174 of 175 patients who were also taking ursodiol to help solubilize the calculous rubble left after treatment. In Boston, of the initial 87 patients accepted, 58 were treated for a single stone (mean diameter 15.7 mm), 16 were treated for 2 stones, and 13 for 3 stones. Although reporting of exact results is not possible before the Food and Drug Administration issues a clearance, it is fair to say that both patients and physicians are generally pleased with the results.

Interventional radiology in the management of bile duct injuries.

The advent of laparoscopic cholecystectomy has brought an increased number of bile duct injuries to the attention of surgeons and interventional radiologists. The spectrum of injury ranges from cystic duct stump leakage to partial obstruction to complete occlusion of the ducts and common hepatic or common bile duct ischemic strictures. The proper therapy for any given patient may be surgical or nonsurgical. Frequently, interventional radiologic techniques permit successful nonoperative drainage of postoperative fluid collections and percutaneous management of bile duct injury and stricture. In some patients in whom radiologic techniques are not successful or in whom the extent of injury requires surgical repair, interventional radiologic techniques can provide preoperative anatomic definition of the extent of injury as well as catheter localization of intrahepatic duct strictures before proximal hepaticojejunostomy.

A theoretical model using mechanical principles to quantify the physical principles of balloon dilatation.

RATIONALE AND OBJECTIVES: Balloon dilatation is a mechanical form of controlled injury used to alleviate vascular stenoses. Several factors influence successful angioplasty. Few mechanical models exist to illustrate the physical principles of balloon dilatation. METHODS: We used mechanical analysis of membrane stresses, along with Laplace's law, to determine a relation between balloon inflation and dilating pressures exerted by balloons in stenoses of varying severity, length, and eccentricity. The balloons were assumed to be perfectly inelastic and flexible. We also examined the resultant stresses in the lesion wall of concentric and eccentric stenoses from exertion of dilating pressures. RESULTS: Dilating pressures depend directly on maximal balloon inflation pressure and balloon diameter. Short, focal stenoses experience greater dilating pressures, which often are several multiples of the inflation pressure, than similarly narrowed longer lesions. CONCLUSION: Dilating pressures depend on inflation pressure, balloon diameter, and lesion severity.

Radiofrequency tissue ablation: increased lesion diameter with a perfusion electrode.

RATIONALE AND OBJECTIVES: We sought to induce large zones of coagulation necrosis using radiofrequency (RF) with perfusion electrodes and to define optimal parameters for this system. METHODS: We developed RF electrodes with internal cannulas to enable tip perfusion. Lesions were created with monopolar RF in ex vivo and in vivo liver and muscle tissue with and without perfusion of the electrode tip using 0 degree C saline. In separate experiments, wattage, current, procedure duration, tip exposure, and perfused tip temperatures were studied. RESULTS: In ex vivo liver tissue, a maximum lesion diameter of 3.1 cm without charring occurred with perfusion at 12 min and 50 W. In in vivo liver tissue with perfusion (tip temperature = 25-35 degrees C) and a 3-cm tip exposure, 80 W were deposited in muscle tissue and 65 W in liver tissue for 12 min without inducing charring. Lesion diameters were 4.5 cm and 2.4 cm, respectively. By comparison, without perfusion a maximum of 20 W could be deposited into either tissue type, resulting in 1.8-cm muscle lesions and 1.2-cm liver lesions. Tip temperatures between 45 degrees C and 55 degrees C resulted in charring. Smaller but predictable lesion diameters were created with a lower power, a shorter tip exposure, or both. Of all the parameters, diameter correlated best with the current applied. CONCLUSION: Perfusion of RF electrodes with chilled saline allows for increased power deposition without tissue charring, increasing the volume of coagulation necrosis created with a single electrode insertion. Perfusion electrodes therefore might decrease the number of probe insertions required for percutaneous tumor ablation therapy or allow for the treatment of larger lesions.