Cloning and heterologous expression of the genes encoding nonspecific electron transport components for a cytochrome P450 system of Saccharopolyspora erythraea involved in erythromycin production.

The forA gene encoding a protein that can function as a NADH:ferredoxin oxidoreductase (For) has been cloned from Saccharopolyspora erythraea, the erythromycin A (ErA) producer. In a previous study For protein, together with the FdxA ferredoxin from the same organism, was shown to be able to reconstitute the cytochrome P450 system responsible for the hydroxylation of 6-deoxyerythronolide B, an intermediate of ErA biosynthesis. Nucleotide sequence data suggest that the cloned forA gene codes for For, the putative pyruvate dehydrogenase component, dihydrolipoamide dehydrogenase, or its close homolog.

Fluorescence lifetime-based sensing in tissues: a computational study.

We have numerically solved the photon diffusion equation to predict the distribution of light in a tissue model system with a uniform concentration of fluorophore. Our results show that time-dependent measurements of light propagation can be used to monitor the fluorescent lifetimes of a uniformly distributed fluorophore in tissues. With proper referencing, frequency-domain measurements of phase-shift, theta, may allow quantitation of fluorescent lifetimes, tau, independent of changes in the local absorption and scattering properties.

Superparamagnetic iron oxide imaging of focal liver disease.

The use of iron oxide as a superparamagnetic contrast agent for magnetic resonance imaging of the liver has been described previously. When administered intravenously, superparamagnetic iron oxide (SPIO) is sequestered by the reticuloendothelial system causing significant shortening of the T2-relaxation time. The majority of the contrast is taken up by the reticuloendothelial tissue of the liver.

A cytochrome P450-like gene possibly involved in oleandomycin biosynthesis by Streptomyces antibioticus.

A cosmid clone from an oleandomycin producer, Streptomyces antibioticus, contains a large open reading frame encoding a type I polyketide synthase subunit and an oleandomycin resistance gene (oleB). Sequencing of a 1.4-kb DNA fragment adjacent to oleB revealed the existence of an open reading frame (oleP) encoding a protein similar to several cytochrome P450 monooxygenases from different sources, including the products of the eryF and eryK genes from Saccharopolyspora erythraea that participate in erythromycin biosynthesis.

Regulation of daunorubicin production in Streptomyces peucetius by the dnrR2 locus.

Sequence analysis of the dnrR2 locus from the cluster of daunorubicin biosynthesis genes in Streptomyces peucetius ATCC 29050 has revealed the presence of two divergently transcribed open reading frames, dnrN and dnrO. The dnrN gene appears to encode a response regulator protein on the basis of conservation of the deduced amino acid sequence relative to those of known response regulators and the properties of the dnrN::aphII mutant. Surprisingly, amino acid substitutions (glutamate and asparagine) at the putative site of phosphorylation (aspartate 55) resulted in a reduction rather than a complete loss of DnrN activity.

Functional characterization and transcriptional analysis of the dnrR1 locus, which controls daunorubicin biosynthesis in Streptomyces peucetius.

We previously proposed that the adjacent dnrIJ genes represent a two-component regulatory system controlling daunorubicin biosynthesis in Streptomyces peucetius on the basis of the homology of the DnrI and DnrJ proteins to other response regulator proteins and the effect of a dnrI::aphII mutation. In the present paper we report the results of work with the dnrI::aphII mutant in complementation, bioconversion, and transcriptional analysis experiments to understand the function of dnrI. For five putative operons in the sequenced portion of the S.

Cloning of the genes encoding thymidine diphosphoglucose 4,6-dehydratase and thymidine diphospho-4-keto-6-deoxyglucose 3,5-epimerase from the erythromycin-producing Saccharopolyspora erythraea.

Genes involved in deoxysugar metabolism, encoding thymidine diphospho (TDP)-glucose 4,6-dehydratase (gdh) and a putative TDP-4-keto-6-deoxyglucose 3,5-epimerase (kde), were cloned from the erythromycin (Er)-producing Saccharopolyspora erythraea by means of an oligodeoxynucleotide corresponding to a segment of the purified Gdh protein. Determination of the nucleotide sequence established that kde lies 3' to gdh. The function of gdh was confirmed by an enzymatic assay following expression of the gene in Escherichia coli.

Off-resonance binomial composite pulses in 2D and 3D fat/water imaging at 0.5 T.

A new method of acquiring two-dimensional (2D) and three-dimensional (3D) fat/water decomposed images on a 0.5 T magnetic resonance (MR) imaging system is described. The method uses a gradient-recalled echo (GRE) sequence incorporating a composite 121 pulse, originally used for magnetization transfer contrast (MTC) experiments, for frequency-selective pre-saturation.

Use of MRI for measuring structures in frozen postmortem brain.

A method was developed for magnetic resonance imaging (MRI) of human autopsy brains stored long-term at -70 degrees C. Scanning brains at temperatures between -70 and -8 degrees C gave minimal MRI signals consistent with protons having limited freedom of movement at low temperature. Raising brain temperature improved the signal such that scanning at -1 degree C generated images with good in-plane resolution, grey/white matter contrast, and fine detail of cortical sulcal/gyral patterns.

Polyketide synthase gene manipulation: a structure-function approach in engineering novel antibiotics.

Polyketides are produced primarily in microorganisms through a specialized metabolism that is a variation of fatty acid biosynthesis. A strong sequence and mechanistic similarity among many of the fatty acid and polyketide synthase enzymes has led to two paradigms for explaining polyketide biochemistry. In one, polyketides are formed by enzyme complexes consisting of four to seven monofunctional proteins in which the beta-carbonyl groups of the intermediates resulting from the condensation of acetate residues are largely not reduced and cyclization of the intermediates typically produces aromatic compounds.

Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens.

The significance of potential active site motifs for acyltransferase and beta-ketoacyl:acyl carrier protein synthase regions within the TcmK protein was investigated by determining the effects of mutations in the proposed active sites on the production of tetracenomycins F2 and C. In a Streptomyces glaucescens tcmGHI JKLMNO null mutant, plasmids carrying the S351A mutation produced high amounts of tetracenomycin F2 but plasmids carrying the C173A or C173S mutation or the H350L-S351A double mutation produced no detectable amount of any known intermediate. In a tcmK mutant, plasmids with the S351A mutation restored high production of tetracenomycin C and plasmids carrying the other mutations were able to complement the chromosomal defect to some extent.

MRI versus arthroscopy in evaluating knee meniscal pathology.

The purpose of this project was to evaluate magnetic resonance imaging (MRI) accuracy for diagnosing and defining meniscal pathology. The charts of 45 patients who underwent both MRI and arthroscopy were viewed retrospectively. All cases were seen by one of two orthopedic surgeons.

Characterization of the Streptomyces peucetius ATCC 29050 genes encoding doxorubicin polyketide synthase.

The dps genes of Streptomyces peucetius, encoding daunorubicin (DNR)-doxorubicin (DXR) polyketide synthase (PKS), are largely within an 8.7-kb region of DNA that has been characterized by Southern analysis, and gene sequencing, mutagenesis and expression experiments. This region contains nine ORFs, many of whose predicted products are homologous to known PKS enzymes.

Triple hydroxylation of tetracenomycin A2 to tetracenomycin C in Streptomyces glaucescens. Overexpression of the tcmG gene in Streptomyces lividans and characterization of the tetracenomycin A2 oxygenase.

Nucleotide sequence analysis of the tcmG gene has suggested that the TcmG protein is responsible for the triple-hydroxylation of tetracenomycin (Tcm) A2 to Tcm C in Streptomyces glaucescens (Decker, H., Motamedi, H., and Hutchinson, C.

Magnetic resonance image synthesis using a flexible model.

Image synthesis methods are based on the hypothesis that a magnetic resonance (MR) image with optimized contrast can be reproduced by synthesis from three calculated basic images of T1, T2 and spin density. This method, however, is limited by noise due to uncertainties in the initial measurements. The principal component analysis (PCA) method is based on an information theory approach that decomposes MR images into a small set of characteristic feature images.

Amino acid catabolism and antibiotic synthesis: valine is a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae.

Targeted inactivation of the valine (branched-chain amino acid) dehydrogenase gene (vdh) was used to study the role of valine catabolism in the production of tylosin in Streptomyces fradiae and spiramycin in Streptomyces ambofaciens. The deduced products of the vdh genes, cloned and sequenced from S. fradiae C373.

Laparoscopic cholecystectomy. Do preoperative factors predict the need to convert to open?

We reviewed our experience with the last 587 laparoscopic cholecystectomies performed between May 1990 and January 1993 to correlate preoperative findings that may predict the conversion of a laparoscopic cholecystectomy to that of an open procedure. The prediction of a need to convert to an open cholecystectomy would allow the surgeon to discuss the higher risk of conversion with the patient and also allow for an earlier intraoperative decision to convert if difficulty was encountered. In addition to routine demographic data, ultrasound reports were available for 526 patients and the following information was recorded: presence of stones, thickened gallbladder wall, common bile duct dilatation, gallbladder sludge, and cystic duct impaction.

Altered clinical presentation of early syphilis in patients with human immunodeficiency virus infection.

Objectives: To compare clinical presentations of patients with early syphilis who did or did not have human immunodeficiency virus (HIV) infection.

Design: Case-control study.

Setting: The sexually transmitted diseases clinics of the Baltimore City Health Department.

Development of a computer workstation for famine early warning and food security.

Beginning in 1990, the University of Arizona, Arizona Remote Sensing Center (ARSC) has been involved in a collaborative effort with the Global Information and Early Warning System (GIEWS) and the Remote Sensing Center of the Food and Agriculture Organization (FAO) of the United Nations in designing and developing an integrated computer workstation for famine early warning. The goal of the project is to provide food security analysts with a set of computer tools to manage a very large and diverse set of data for predicting the onset of food security emergencies for every country on Earth. The initial stage of the project involved the conceptual definition of system elements and the development of overall system architecture.

The genetic basis of precursor supply for the biosynthesis of macrolide and polyether antibiotics.

Macrolide and polyether biosynthesis in actinomycetes is regulated at the level of precursor supply by effects of nutrients on the sources of the low-molecular-weight fatty acids used to build the carbon framework of these antibiotics. Ammonium ion appears to suppress the first enzymes of valine and threonine catabolism and also inhibits their activity. Disruption of the valine dehydrogenase (vdh) gene of Streptomyces coelicolor destroys its ability to grow on branched-chain amino acids as the sole nitrogen source in a minimal medium but has no effect on the biosynthesis of the acetate-derived antibiotic, actinorhodin.

Clinical evaluation of fast spin echo sequences for cranial magnetic resonance imaging at 0.5 tesla.

Fast spin echo (FSE) sequences enable T2-weighted MR scans to be obtained in a fraction of the time necessary for conventional SE sequences with long TR and long TE. Comparison has been made of a FSE sequence (TR = 4000 ms, effective TE = 100 ms, 2 NEX) with the T2-weighted SE sequence (TR = 2000 ms, TE = 90 ms, 1-2 NEX) normally used in 35 patients referred for cranial MRI. Contrast-to-noise ratios (CNR) for grey:white matter and brain:CSF on the FSE sequence compared favourably with variable echo (VE) sequences which take up to three times as long to acquire.