Structural insights into the ene-reductase synthesis of profens.

Reduction of double bonds of α,ß-unsaturated carboxylic acids and esters by ene-reductases remains challenging and it typically requires activation by a second electron-withdrawing moiety, such as a halide or second carboxylate group. We showed that profen precursors, 2-arylpropenoic acids and their esters, were efficiently reduced by Old Yellow Enzymes (OYEs). The XenA and GYE enzymes showed activity towards acids, while a wider range of enzymes were active towards the equivalent methyl esters. Comparative co-crystal structural analysis of profen-bound OYEs highlighted key interactions important in determining substrate binding in a catalytically active conformation. The general utility of ene reductases for the synthesis of (R)-profens was established and this work will now drive future mutagenesis studies to screen for the production of pharmaceutically-active (S)-profens.

Anaesthetic management of a patient with idiopathic pulmonary arterial hypertension, suprasystemic pulmonary artery pressures and carcinoma of the ascending colon.

A 35-year-old woman with severe pulmonary arterial hypertension underwent open hemicolectomy with cholecystectomy under combined general and epidural anaesthesia. Intra-operative pulmonary artery pressure, as measured by Swan-Ganz catheter, was suprasystemic and managed with inodilators. She developed postoperative right ventricular dysfunction requiring inotropes, incremental pulmonary vasodilators and prolonged oxygen supplementation. One year after surgery, she is recurrence-free with oxygen saturations of 88-90% on air. This case highlights that with meticulous care and multidisciplinary team input, patients with severe pulmonary arterial hypertension can have favourable outcomes after major cancer surgery.

Replitase: a complex integrating dNTP synthesis and DNA replication.

Replitase is a multienzyme complex of mammalian cells that produces deoxynucleoside triphosphates and delivers them to the DNA polymerase activity, which also resides in the complex. Structural interactions within this complex form the basis of internal controls to keep these key biosynthetic processes efficient and in balance. The active complex is formed in the nuclear region in only the S phase of the cell cycle, when the cell's DNA is being replicated. Replitase is a member of the growing family of structured, multienzyme, biosynthetic complexes for which very similar complexes are found in prokaryotes and eukaryotes. Logically, the most basic of all biosynthetic pathways should show the efficiency and precise controls that even lesser pathways possess and, in fact, this seems to be so. In this article, we have outlined a broad range of evidence supporting the existence of the replitase complex in mammalian cells, a complex for dNTP synthesis and polymerase that exists only in the S phase and only in the nuclear region. This is consistent with localization studies in intact cells and after various forms of cell fractionation and, particularly, with experiments of incorporation of precursors into DNA in isolated complexes and S phase permeabilized cells. A most forceful argument for replitase is the existence of three striking phenomena--channeling, compartmentation, and cross-inhibition. These are very difficult, perhaps impossible, to explain without replitase; with replitase, their explanation is beautifully simple.

Esophageal compression by the aorta after arterial switch.

Extrinsic compression of the esophagus in children most often occurs in the presence of a congenital vascular ring. We recently operated on a patient in whom esophageal compression had developed that was severe enough to require feeding via a gastrostomy tube several years after the arterial switch operation. Aortopexy and extensive mediastinal mobilization were performed twice with transient relief and gradual return of symptoms. Almost 3 years after the first aortopexy, lasting relief was achieved by transposing the esophagus into the right side of the chest.

Relative role of genes and environment on BP: twin studies in Madras, India.

This study was conducted to test the feasibility of the twin research model in a developing country with diverse cultures and to understand the relative influence of genetic and environmental factors on BP variation among South Indians. This was a cross-sectional twin study of volunteers using a two-by-two factorial design for the analysis of quantitative traits. The factors were twin type (monozygotic and dizygotic) and sex (male and female). The study was conducted in Madras. Twenty-four pairs of twins were contacted for participation in the project. Of the 24 pairs we contacted, 91% (20) actually participated in our study. Among 20 sets we studied, 10 (50%) are males and 10 (50%) are females with an average age of 23 years. The mean SBP of this volunteer twin population was 115.18 +/- 1.27 mmHg and DBP was 68.53 +/- 1.41 mmHg. Analysis of dietary habits (vegetarian/nonvegetarian) showed that BP was greater (118.26 +/- 2.29/71.88 +/- 2.34 mmHg) in vegetarian twins than nonvegetarians (112.28 +/- 1.42/66.2 +/- 1.90 mmHg). Also a positive correlation between urinary excretion of calcium and BP was observed. The present study demonstrates that epidemiological research in a developing country like India is feasible and economical, using the twin research methodology. As observed in other populations, the major source of BP variation in the population appears to be predominantly under genetic control.

Lipid profiles among fish-consuming coastal and non-fish-consuming inland populations.

Serum lipid profiles were examined in 100 individuals of fish-consuming coastal and 109 individuals of non-fish-consuming inland populations. The fish-consuming population showed lower mean serum cholesterol, triacylglycerols and significantly higher levels of HDL cholesterol and phospholipids. The bleeding time and clotting time were significantly prolonged in the fish-eating population. Thus the fish-consuming population showed lower risk factors of coronary heart disease when compared to the non-fish-consuming population.

Structure of a muramic acid containing capsular polysaccharide from the pathogenic strain of Vibrio vulnificus ATCC 27562.

Vibrio vulnificus strains isolated from septicemia cases and from the environment show a wide variety of capsular types. In an attempt to find common structural features which can be correlated with pathogenicity and toxicity, we have determined structures of the capsular polysaccharides (CPS) from several pathogenic strains. We report the complete structure of the polysaccharide from the pathogenic V. vulnificus strain ATCC 27562 using a combination of homonuclear and heteronuclear one-dimensional and two dimensional NMR experiments. The 13C and 1H NMR spectra, including the exchangeable amide proton resonances, have been completely assigned. The amide linkage between Ser and C6 of GalA has been unambiguously determined by water-suppressed 2D NOESY. To verify the structure established by NMR, we have fragmented the polymer employing the Smith degradation procedure. The Smith product identified by NMR and matrix-assisted laser desorption mass spectrometry is consistent with the proposed structure for the CPS, which is composed of D-GlcNAc, MurNAc, D-GalA, L-Rha and is serine-linked as shown: [formula: see text]

MR imaging of the thoracic aorta.

MR imaging is an effective modality for noninvasive morphologic and functional assessment of the thoracic aorta. MR imaging provides several advantages for vascular imaging, including intrinsic contrast between the blood pool and vascular structures, multiplanar imaging capability, and the absence of ionizing radiation. By combining imaging and flow-sensitive techniques, MR imaging can be used to delineate morphology and to quantify bloodflow volume and velocity. In patients who need sequential examinations over time to monitor disease severity, a noninvasive technique such as MR imaging is desirable.

Aneurysm of saphenous vein coronary bypass graft: diagnosis by computed tomography.

A patient with a history of coronary artery bypass graft surgery underwent computed tomography scanning for evaluation of a lung mass. A heterogeneous mediastinal mass discovered incidentally on computed tomography scanning was shown to be a saphenous vein bypass graft aneurysm.

Low-dose, dynamic, expiratory thin-section CT of the lungs using a spiral CT scanner.

Seventeen patients with lung transplants were evaluated with inspiratory, postexpiratory, and low-dose, dynamic expiratory thin-section computed tomography (CT). Region of interest measurements were performed on inspiration and expiration images with both techniques, and mean lung attenuation changes between inspiration and expiration images were calculated and compared. Dynamic expiratory thin-section CT resulted in a significantly greater increase in lung attenuation than postexpiratory thin-section CT. Dynamic expiratory thin-section CT may prove useful in the evaluation of patients with lung diseases characterized by air flow obstruction with little increase in patient radiation dose.

Incidental detection of thoracic sarcoidosis on whole-body 18fluorine-2- fluoro-2-deoxy-D-glucose positron emission tomography.

18 Fluorine-2- Fluoro-2-Deoxy-D-Glucose positron emission tomography (18FDG PET) allows imaging of sites with increased metabolic activity. Increased metabolic activity in mediastinal nodes in sarcoidosis has been described. We report the prospective diagnosis of thoracic sarcoidosis on 18FDG PET based on extensive, peripheral, upper lobe parenchymal, and mediastinal nodal tracer uptake.

Congenital heart disease: measuring physiology with MRI.

Cine MRI and VEC MRI can be used to quantitate the physiology of the heart and great vessels in patients with CHD. This information can be a valuable adjunct to anatomical imaging for preoperative planning as well as postoperative monitoring. Some important clinical applications of quantitative cardiovascular functional MRI include measurement of ventricular masses, stroke volumes, and ejection fractions; estimation of shunts and valvular regurgitation; assessment of collateral blood flow and pressure gradients in aortic coarctation; and postsurgical evaluation of conduit blood flow and pressure gradients.

Imaging evaluation of suspected pulmonary embolism.

Venous thromboembolism (VTE) is a common disorder that is difficult to diagnose clinically but carries significant morbidity and mortality if untreated. Additionally, although demonstrated to be of benefit in cases of proven deep vein thrombosis (DVT) and pulmonary embolism (PE), anticoagulation therapy is not without risk. Because the clinical exam is known to be unreliable for the detection of both DVT and PE, many imaging modalities have been used in the diagnostic imaging algorithm for the detection of VTE, including chest radiography, ventilation/perfusion (V/Q) scintigraphy, pulmonary angiography, and recently, spiral computed tomography (CT) and magnetic resonance imaging (MRI). Chest radiographic findings in acute PE include focal oligemia, vascular enlargement, atelectasis, pleural effusions, and air space opacities representing pulmonary hemorrhage or infarction. The chest radiograph can occasionally be suggestive of PE but is more often nonspecifically abnormal. The main use of the chest radiograph in the evaluation of suspected PE is to exclude entities that may simulate PE and to assist in the interpretation of V/Q scintigraphy. Lower extremity venous compression ultrasonography (CU) is both sensitive and specific for the diagnosis of femoropopliteal DVT, and the value of negative CU results has been established in outcomes studies. However, the reliability of CU for the detection of isolated calf vein thrombosis is not well established, and the clinical significance of such thrombi is debatable. Additional methods such as color and spectral Doppler analysis are also useful in the diagnostic evaluation of DVT but are best considered as adjuncts to the conventional CU examination rather than as primary diagnostic modalities themselves. Compression ultrasonography and Doppler techniques are useful in the evaluation of suspected upper extremity DVT; spectral Doppler waveform analysis is particularly useful to assess for the patency of veins that cannot be directly visualized and compressed with conventional gray-scale sonography. V/Q scintigraphy has been the initial modality obtained in patients suspected of PE for a number of years. Although many studies have investigated the role of V/Q scintigraphy in the evaluation of VTE, the Prospective Investigation of Pulmonary Embolism Diagnosis (PIOPED) study has provided the most useful information regarding the utility of V/Q scintigraphy in this setting. A high probability scan interpretation is sufficient justification to institute anticoagulation, and a normal perfusion scan effectively excludes the diagnosis of PE. A normal/near normal scan interpretation also carries a sufficiently low prevalence of angiographically proven PE to withhold anticoagulation. Although the prevalence of PE in the setting of low probability scan interpretations is low and several outcomes studies have demonstrated a benign course in untreated patients with low probability scan results, patients with inadequate cardiopulmonary reserve do not necessarily have good outcomes. Such patients deserve more aggressive evaluation. Patients with intermediate probability scan results have a 20% to 40% prevalence of angiographically proven PE and thus require further investigation. The radionuclide investigation of DVT includes such techniques as radionuclide venography and thrombus-avid scintigraphy. Although these methods have not been as thoroughly evaluated as CU, studies thus far have indicated encouraging results, and further investigations are warranted. Pulmonary angiography has been the gold standard for the diagnosis of PE for decades. Studies have indicated that angiography has probably been underutilized by referring physicians for the evaluation of suspected PE, likely because of the perception of significant morbidity and mortality associated with the procedure. (ABSTRACT TRUNCATED)

Compartmentation of deoxypyrimidine nucleotides for nuclear DNA replication in S phase mammalian cells.

DNA synthesis in S phase Chinese hamster embryo fibroblast cells in the presence of exogenous 3H-dUrd shows incorporation of the labeled precursor with very little dilution by the large unlabeled intracellular precursor pools. Full mixing would predict a specific activity 10-fold less than that measured. This coupled with the finding that 80% of the radioactivity derived from the exogenous 3H-dUrd appears in the karyoplasts implies a compartmentation where 3H-dUMP and 3H-dTTP derived from exogenous 3H-dUrd do not mix freely with endogenous cytoplasmic pools.

Cell cycle: regulatory events in G1-->S transition of mammalian cells.

A cell divides into two daughter cells by progressing serially through the precisely controlled G1, S, G2, and M phases of the cell cycle. The crossing of the G1/S border, which is marked by the initiation of DNA synthesis, represents commitment to division into two complete cells. Beyond this critical point no further external signals are required. We now have more comprehensive knowledge of the temporal sequence of systems at this key transition from G1 to S--growth factor responses, a cascade of kinase reactions, activation of cyclins and their associated kinases, and oncogene and tumor suppressor gene products. Furthermore, we know that the absolute requirement for calcium and the timing of events associated with calmodulin and the 68 kDa calmodulin-binding protein are consistent with overall Ca++/calmodulin control of all steps from the response to growth factors in G1 to DNA replication in S phase. We now have to sort out the inter-relationships of myriad control proteins and their relation to the Ca++/calmodulin-dependent controls--Which are causes? Which are effects? And which are parallel processes? The answers will be important, as they represent both a much deeper understanding of this key process of life and an important opportunity for improving therapeutic medicine.

Thin layer chromatography of dansyl and dinitrophenyl derivatives of amino acids. A review.

The dinitrophenyl (DNP) derivatives of amino acids have found continual application in protein sequencing since Sanger used them for the first time for the sequencing of insulin. Dansyl derivatives of amino acids have been widely used in protein sequencing because of their fluorescent nature. The success of protein sequencing largely depends upon correct identification of such derivatives. The choice for the method of identification is related to cost, the availability of instrumentation and to the sensitivity needed for the analysis. Thin layer chromatography (TLC) is simple and has several advantages over other chromatographic methods. Therefore the literature after 1972 is reviewed for TLC analysis of dansyl- and DNP-amino acids, the two important amino acid derivatives required for identifying protein sequences. Additionally, the literature on the TLC resolution of enantiomeric mixtures of dansyl amino acids is reviewed. Application of various adsorbents, composition of solvent systems and other experimental conditions together with successful resolution data have been discussed. TLC provides a direct and inexpensive method for the resolution of enantiomers, and is fast becoming a sensitive instrumentalized quantitative analytical technique.