Peroxisomal multifunctional enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization.

In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol.

Peroxisomal beta-oxidation and polyunsaturated fatty acids.

Peroxisomes are capable of oxidizing a variety of substrates including (poly)unsaturated enoyl-CoA esters. The beta-oxidation of unsaturated enoyl-CoA esters in peroxisomes, and also in mitochondria, is not just chain-shortening but also involves the metabolizing of pre-existing carbon-to-carbon double bonds. In addition to the enzymes of the beta-oxidation spiral itself, this metabolism requires the participation of auxiliary enzymes: delta 3, delta 2-enoyl-CoA isomerase; 2,4-dienoyl-CoA reductase; 2-enoyl-CoA hydratase 2 or 3-hydroxyacyl-CoA epimerase; and delta 3,5 delta 2,4-dienoyl-CoA isomerase.

Expression of mRNAs for neurotrophins and their receptors in developing rat heart.

Because the neurotrophic system has not been systematically studied in developing heart, we studied the expression of mRNAs for neurotrophins and their high- and low-affinity receptors by radioactive in situ hybridization in the rat heart from embryonic day 9 (E9) to parturition. The neurotrophin-3 (NT-3) transcripts were seen in the group of Leu-7 immunoreactive cells in the ventricular region from E11 to parturition, suggesting that NT-3 is expressed in the part of the developing conduction system, mRNAs for truncated trk receptors, trkC.TK- and trkB.

Crystal structure of enoyl-coenzyme A (CoA) hydratase at 2.5 angstroms resolution: a spiral fold defines the CoA-binding pocket.

The crystal structure of rat liver mitochondrial enoyl-coenzyme A (CoA) hydratase complexed with the potent inhibitor acetoacetyl-CoA has been refined at 2.5 angstroms resolution. This enzyme catalyses the reversible addition of water to alpha,beta-unsaturated enoyl-CoA thioesters, with nearly diffusion-controlled reaction rates for the best substrates.

Characterization and isolation of enzymes that hydrolyze short-chain acyl-CoA in rat-liver mitochondria.

In this study we investigated the presence of short-chain acyl-CoA hydrolases in rat liver mitochondria. One acetyl-CoA-hydrolyzing enzyme with a molecular mass of about 48 kDa was purified to apparent homogeneity as judged by SDS/PAGE. Immunoprecipitation experiments with antibodies raised to the purified protein showed that this enzyme corresponds to a minor portion of the total mitochondrial acetyl-CoA hydrolase activity, most (about 90%) of the total activity being due to an enzyme which was labile and required Triton X-100 for its stability.

Initial human studies with single-photon emission tomography using iodine-123 labelled 3-(5-cyclopropyl-1,2,4-oxadiazo-3-yl)- 7-iodo-5, 6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]-benzodiazepine (NNC 13-8241).

The iodine-123 labelled ligand 3-(5-cyclopropyl-1,2, 4-oxadiazo-3-yl)-7-iodo-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1, 5-a][1,4]-benzodiazepine ([123I]NNC 13-8241) was evaluated as a probe for in vivo imaging of benzodiazepine receptor sites in the human brain. Four healthy volunteers were imaged with a high-resolution single-photon emission tomography (SPET) scanner. The metabolism of [123I]NNC 13-8241 in plasma was slow.

Localization of glial cell line-derived neurotrophic factor (GDNF) mRNA in embryonic rat by in situ hybridization.

The localization of glial cell line-derived neurotrophic factor (GDNF) mRNA was studied by in situ hybridization in rat from embryonic (E) day E10 to E15. At E10, GDNF mRNA is found in the urogenital field and the cranial part of the gut. At E11, the most abundant expression of GDNF mRNA is seen in the epithelial cells of the second, third and fourth pharyngeal pouches, the third and fourth pharyngeal arches and pharynx.

Development of 123I-labelled NNC 13-8241 as a radioligand for SPECT visualization of benzodiazepine receptor binding.

[125I]- and [123I]NNC 13-8241 were prepared from the trimethyltin precursor and radioactive iodide using the chloramine-T method. The total radiochemical yields of [125I]- and [123I]NNC 13-8241 were 60-70% and 40-50% respectively, with radiochemical purity higher than 98%. In binding studies with [125I]NNC 13-8241 in rats in vitro and in vivo a high uptake of radioactivity was demonstrated in brain regions known to have a high density of benzodiazepine (BZ) receptors such as the occipital and frontal cortex.

Changing stereochemistry for a metabolic pathway in vivo. Experiments with the peroxisomal beta-oxidation in yeast.

The biosphere is inherently built of chiral molecules, and once their metabolism is established, the stereochemical course of the reactions involved is seen to remain highly conserved. However, by replacing the yeast peroxisomal multifunctional enzyme (MFE), which catalyzes the second and third reactions of beta-oxidation of fatty acids via D-3-hydroxyacyl-CoA intermediates, with rat peroxisomal MFE, which catalyzes the same reactions via L-3-hydroxy intermediates, it was possible to change the chiralities of the intermediates in a major metabolic pathway in vivo. Both stereochemical alternatives allowed the yeast cells to grow on oleic acid, implying that when the beta-oxidation pathways evolved, the overall function was the determining factor for the acquisition of MFEs and not the stereospecificities of the reactions themselves.

Iodine-123 labelled N-(2-fluoroethyl)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)nortropane for dopamine transporter imaging in the living human brain.

There are several cocaine analogs which have potential for imaging the dopamine transporters (DAT). Earlier studies have shown that iodine-123 labelled 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane ([123I]beta-CIT) and N-(3-fluoropropyl)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)nortropane ([123I]beta-CIT-FP) are promising DAT imaging agents in the living human brain with single-photon emission tomography (SPET). Here we report a pilot comparison of [123I]beta-CIT and [123I]beta-CIT-FP with a new tropane derivative, [123I]N-(2-fluoroethyl)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)nortropane ([123I]beta-CIT-FE), using SPET imaging in four healthy male subjects.

[191mIr: distribution and retention in animal experiments].

Ultrashort-lived tracer 191mIr (T1/2 = 4.92 s) can be obtained with a high yield from an 191Os/191mIr generator with a low 191Os breakthrough. It was eluated directly into the tail veins of Wistar rats.

Very long chain and long chain acyl-CoA thioesterases in rat liver mitochondria. Identification, purification, characterization, and induction by peroxisome proliferators.

We have previously reported that long chain acyl-CoA thioesterase activity was induced about 10-fold in rat liver mitochondria, when treating rats with the peroxisome proliferator di(2-ethylhexyl)phthalate (Wilcke M., and Alexson S. E.

Lipophilic metabolite of [123I]beta-CIT in human plasma may obstruct quantitation of the dopamine transporter.

I-123 or C-11 labelled 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane (beta-CIT) is a recently developed radioligand for the study of dopamine and serotonin reuptake sites in humans with single photon emission tomography (SPET) or positron emission tomography (PET). Determination of the radioligand metabolite pattern is fundamental for a quantitative analysis of radioligand binding. The metabolism of [123I]beta-CIT was determined by a gradient HPLC method in plasma samples of six human subjects.

Comparison of iodine-123 labelled 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane and 2 beta-carbomethoxy-3 beta-(4-iodophenyl)-N-(3-fluoropropyl)nortropane for imaging of the dopamine transporter in the living human brain.

Several cocaine congeners are of potential for imaging the dopamine transporter (DAT). Previous studies have shown that iodine-123 labelled 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane ([123I] beta-CIT) is a promising radiotracer for imaging the serotonin (5-HT) and dopamine (DA) transporters in the living human brain with single-photon emission tomography (SPET). [123I] beta-CIT was found to be not very practical for 1-day DAT imaging protocols since peak DAT uptake occurs later than 8 h.

Accumulation of 99mTc-low-density lipoprotein in human malignant glioma.

Low-density lipoprotein (LDL) uptake in gliomas was studied to find out if LDL has potential as a drug carrier of boron, especially for boron neutron capture therapy. Single photon emission tomography (SPET) was performed 2 h and 20 h after intravenous injection of autologous 99mTc-labelled LDL in four patients with untreated and five patients with recurrent glioma. 99mTc-LDL uptake was compared with the uptake of 99mTc-labelled human serum albumin (HSA), an established blood pool marker.

Isolation and characterization of cDNA for human 120 kDa mitochondrial 2,4-dienoyl-coenzyme A reductase.

2,4-Dienoyl-CoA reductase (EC 1.3.1.

191Os/191mIr-generator for studying arterial blood flow in experimental animal models.

Iridium-191m (191mIr, t1/2 = 4.96 sec), an ultra-short lived tracer, has turned out to be suitable for gamma imaging. It can be obtained in high yields from an 191Os/191mIr-generator with a low 191Os breakthrough.

A low pH 111In-bleomycin complex: a tracer for radiochemotherapy of head and neck cancer.

Bleomycin (BLM) is a well known natural antibiotic. It is toxic to dividing cells and has been used for the treatment of several forms of cancer. BLM has been labeled with various cations, but most of them have turned out be unstable in in-vivo experiments.

Samarium-153-EDTMP in bone metastases.

Thirty-five patients with painful bone metastases arising from a variety of tumor types underwent a clinical trial in which 153Sm-EDTMP was injected as a single intravenous dose. The injection ranged in amount from 330 MBq to 1110 MBq of 153Sm-EDTMP. Pain relief usually occurred within one week after administration.

Mitochondrial 2,4-dienoyl-CoA reductases in the rat: differential responses to clofibrate treatment.

The effect of a peroxisome proliferator, clofibrate, on mitochondrial 2,4-dienoyl-CoA reductases was studied in rat liver. The specific activity of reductase measured with 2,4-hexadienoyl-CoA as the substrate increased 2.9-fold in the liver homogenate and 2.

Crystallization experiments with 2-enoyl-CoA hydratase, using an automated 'fast-screening' crystallization protocol.

A convenient method for screening crystallization conditions using an automated fast-screen protocol has been implemented and tested on an enoyl-CoA hydratase. The crystallization solutions for the initial screening and subsequent optimizations are prepared using a crystallization robot. Enoyl-CoA hydratase (E.

cDNA cloning and amino acid sequence of human mitochondrial delta 3 delta 2-enoyl-CoA isomerase: comparison of the human enzyme with its rat counterpart, mitochondrial short-chain isomerase.

We report the isolation of a cDNA encoding a mature human monofunctional delta 3 delta 2-enoyl-CoA isomerase and the determination of its nucleotide sequence. The purified uncleaved protein, as well as several internal tryptic and CNBr fragments, were subjected to N-terminal peptide sequencing. The deduced amino acid sequence of the mature protein consists of 260 amino acids with a predicted M(r) of 28735.

Classification of the reflectance spectra of pine, spruce, and birch.

Statistical pattern-recognition methods are applied to the classification of the reflectance spectra of growing trees (Scots pine, Norway spruce, and birch). We show by using large training sets that it is possible to develop classification filters that are able to discriminate the tree types with a very high probability. Our approach may offer a reference coordinate system for multispectral remote sensing of different levels of forest damage.