Synthesis and evaluation of fluorine-18 labelled compounds for imaging of bacterial infections with pet.

Syntheses of no carrier added (n.c.a.) 6-fluoro-1,4-dihydro-1-cyclopropyl-4-oxo-7-[4-[18F]fluoro-phenacyl-1-piperacinyl]-chinolincarboxylic acid ([18F]COPCA) and n.c.a. 4-[18F]fluoro-benzoyl-ubiquicidin 29-41 ([18F]UBI 29-41) are described. [18F]COPCA was synthesised within 120 min with a radiochemical yield of 9-12%. [18F]UBI 29-41 was synthesised within 150 min with a radiochemical yield of 15-20%. Both compounds had a specific activity of more than 35 GBq/micromol. The biological activity was verified by measuring its binding to Staphylococcus aureus bacteria. Specific binding was found for [18F]UBI 29-41 (12-17%), whereas no specific binding for [18F]COPCA was found.

Synthesis and evaluation of a 18F-labelled recombinant annexin-V derivative, for identification and quantification of apoptotic cells with PET.

In this report, we describe the synthesis of 4-[18F]-fluorobenzoyl-annexin V (4-[18F]FBA). In a four-step procedure, 4-[18F]FBA was synthesised with a microcomputer controlled, automated module within 90min. The radiochemical yield was in the range of 15-20% (corrected for decay) with a specific activity of more than 35GBq/micromol. The specific binding was confirmed by studies of 4-[18F]FBA with phosphatidylserine-containing liposomes. The biological activity of 4-[18F]FBA was verified by measuring its binding to Jurkat T-cell lymphoblasts after induction of apoptosis as compared to control cells without apoptosis. 4-[18F]FBA seems to be a suited tracer to measure apoptotic activity in vivo.

Malignant ascites fluid (MAF), including ovarian-cancer-associated MAF, contains angiostatin and other factor(s) which inhibit angiogenesis.

OBJECTIVE: The aim was to determine whether human malignant ascites fluid (MAF) associated with abdominal cancer, including ovarian cancer, contained factors which inhibit angiogenesis as well as others which stimulate this process. METHODS: MAF was collected from six patients, four with ovarian cancer, one with gastric cancer, and one with liver metastases. Using the chick chorioallantoic membrane (CAM) the effect of MAF on 7-day-old CAM capillaries was examined for 48 h. Vascular endothelial growth factor (VEGF) was evaluated by ELISA. Five samples of MAF were fractionated by lysine-Sepharose chromatography and the lysine-bound and -unbound fractions were eluted by epsilon-amino-n-hexanoic acid. Whole MAF, the lysine-bound and -unbound fractions, and human angiostatin were subjected to SDS-PAGE/Western blot analysis and immunostained after exposure to anti-human plasminogen. Human plasminogen was exposed to conditioned medium from ovarian epithelial cancer (HEY) cells and subjected to similar Western blot analysis. RESULTS: Despite containing VEGF, each MAF sample examined caused a loss of capillaries from the CAM; a similar response was seen using purified human angiostatin. Whole MAF and the lysine-bound fraction contained plasminogen (90 kDa) and a 55-kDa protein which migrated in a similar manner to human angiostatin on Western blot. Both the lysine-bound and -unbound fractions caused a loss of capillaries in the CAM. Human plasminogen exposed to conditioned medium from HEY cells yielded a fragment which was similar in size to angiostatin. CONCLUSIONS: MAF from patients with various clinical presentations contains angiostatin and VEGF as well as other factors which are capable of inhibiting angiogenesis.

The pattern of micro-organisms and the efficacy of new macrolide in acute lower respiratory tract infections.

Lower respiratory tract infection (LRTI) is one of the major health problems in developing countries such as Indonesia. According to the National Household Health Survey conducted by the Ministry of Health in 1992, LRTIs still rank fourth as the main cause of death in Indonesia. The problem of LRTIs could be simply managed as long as the causative organism can be identified and the proper antibiotic known. In some occasions, it is not quite so easy to identify the causative micro-organism, especially in lower tract infections. There are several methods of obtaining specimens from LRTIs for cultures. The easiest, most simple way is to collect expectorated sputum. Unfortunately, because of the high rate of contamination by upper respiratory tract flora, this method is not reliable. Recognizing the difficulties with routine expectorated sputum cultures, two alternative approaches have been suggested. One approach is to bypass potential expectorated sputum 'contaminants' in the oropharynx by transtracheal aspiration or transthoracic aspiration. The second approach is to modify the usual technique of processing expectorated sputum by either washing techniques or by quantitative cultures. Azithromycin and clarithromycin are chemically related to macrolide erithromycin. Both antibiotics retain the traditional macrolide spectrum of activity against gram-positive and atypical pneumonia pathogens, while demonstrating improved activity against gram-negative bacteria. The American Thoracic Society (ATS) recommended the use of macrolide for outpatients with community-acquired pneumonia, without comorbidity and 60 years of age or younger. A total of 34 outpatients with acute LRTIs were open-comparative, randomly allocated to treatment with the new macrolide in Persahabatan Hospital, Jakarta, 1996. The purposes of this study were: (i) to identify the causative micro-organisms; and (ii) to evaluate the clinical efficacy of the new macrolide in these infections. Azithromycin 500 mg was given orally once a day for 3 days and was administered 1 h before or 2 h after every meal. Clarithromycin 500 mg was given orally every 12 h for 10 days. The diagnosis of the patients were: 16 with pneumonia, 10 with acute bronchitis and 8 with acute exacerbation of chronic bronchitis. In this study of 34 patients, the sputum specimens were washed with N acetylcysteine before culture and we could only detect micro-organisms in one patient. Before treatment, we found 47 strains in 33 (97.05%) patients and after treatment we found five strains. From serological examination, only four (11.76%) atypical bacterial were detected. The most frequently found microorganisms were 23 strains of Klebsiella pneumoniae (40.42%), 10 of Streptococcus alpha haemolyticus (21.26%), five of Streptococcus pneumoniae (10.63%) and five of Staphylococcus aureus (10.63%). The atypical bacterial were: two Legionella pneumophila, one Mycoplasma pneumoniae and one Chlamydia pneumoniae. The clinical efficacy of new macrolides were 100% and the bacteriological responses with eradication of 94.12% vs 70.59% of isolates in the azithromycin and clarithromycin groups are shown in Table 1. There were no adverse reactions detected in the two treatment groups until the end of the study.

Identification and characterization of a monoclonal antibody to the membrane fatty acid binding protein.

A monoclonal antibody to the rat liver membrane fatty acid binding protein (MFABP) was prepared by immunizing mice with purified MFABP isolated from solubilized rat liver plasma membrane proteins by oleate-agarose affinity chromatography technique. The monoclonal antibody K15/6 identified a single 40 kDa protein in rat liver plasma membranes with pI values of 8.5, 8.8 and 9.0, which is identical to the authentic MFABP, but clearly distinct from rat mitochondrial GOT. The antibody K15/6 selectively inhibited cellular influx as well as membrane binding of fatty acids, but not of cholesterol or vitamin E. The same antibody was used in immunofluorescence, ELISA and Western blot analysis to determine the subcellular and organ distribution pattern of MFABP. The protein was identified in rat liver plasma membranes and mitochondria, but in no other cell compartment. It was detectable in homogenates of rat liver but not in homogenates of other organs. Therefore, the monoclonal antibody K15/6 represents an organ specific antibody to MFABP which reveals inhibitory action on membrane binding/transport of fatty acids.

Production of monoclonal antibodies against the purified glycosylphosphatidylinositol anchor of the variant surface glycoprotein from Trypanosoma brucei brucei.

The glycosylphosphatidylinositol anchor (GPI) from the membrane form variant surface glycoprotein (mfVSG) of Trypanosoma brucei brucei was isolated and identified after radioactive labeling with [3H]myristic acid, by immunostaining on HPTLC with a polyclonal antibody directed against mfVSG and by negative ion laser desorption and fast atom bombardment mass spectrometry of the GPI anchor before and after peracetylation. For the production of monoclonal antibodies the purified GPI molecule was incorporated into liposomes and injected intrasplenically in BALB/c mice. After fusion with the myeloma cell line X63-Ag 8.653 hybridoma cells were cloned by single cell cloning. The secreted antibodies were characterized by ELISA, Ouchterlony immunodiffusion, and Western blot and used in first immunofluorescent studies.

Kinetic studies of alkyl-dihydroxyacetone-phosphate (alkyl-glycerone-phosphate) synthase in peroxisomes of rat liver.

The properties of peroxisomal enzyme alkylglycerone-phosphate synthase were studied in highly purified peroxisome fractions of rat liver. The requirements for optimal enzyme activity: pH and composition of the reaction mixture, incubation time, and enzyme concentration were investigated, and kinetic studies performed employing both different long-chain fatty alcohols and acyl dihydroxyacetone phosphates as substrates. Activities of the synthase considerably higher as reported before were found in the peroxisome preparation, with alkylglycerone (alkyldihydroxyacetone) phosphate as the sole product of the exchange reaction. The kinetic studies revealed divergent properties of peroxisomal synthase with respect to the substrates involved. Whereas the substrate concentration versus reaction velocity plot for the fatty alcohols reflects Michaelis-Menten kinetic behavior, it displays a maximum followed by inhibition with regard to the acylglycerone phosphate. The enzyme accepts different acylglycerone phosphates without much specificity but it is most active with 9-cis-octadecenol.

MS and NMR analysis of the cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 variant specific glycoprotein.

The cross-reacting determinant glycan from Trypanosoma brucei brucei MITat 1.6 is known to contain galactose, mannose and non-acetylated glucosamine. The structural elucidation of this oligosaccharide has been impeded by an unusual non-glycosidic linkage to the peptide chain and a glycosidic linkage to inositol phosphate on either side of the oligosaccharide. Using two different approaches for the isolation of the glycan, namely hydrolysis to give the oligosaccharide directly or pronase digestion to yield the glycan-containing C-terminal glycophosphopeptide, the structure of this glycan was elucidated by mass spectrometry and 1H-NMR spectroscopy. There was evidence of heterogeneity in the glycan residue.

Studies on the phase transitions of lysoderivatives of ethanolamine glycerophospholipids from human brain.

The phase transition temperature of 1,2-distearoylglycerophosphocholine is reduced in presence of equimolar amounts of 1-O-(1'-alkenyl)-glycerophosphoethanolamine (ethanolamine lysoplasmalogen) from 53.3 degrees C-54.1 degrees C to 44.0 degrees C-44.9 degrees C at different pH (4.0; 7.2; 9.0; 10.5). 1-Acyl-glycerophosphoethanolamine leads to a smaller reduction of the 1,2-distearoyl-glycerophosphocholine transition temperature: 45.0 degrees C-46.2 degrees C at the same pH-values. 1-Alkyl-glycerophosphoethanolamine (hydrogenated ethanolamine lysoplasmalogen) possesses a transition temperature, which is 3.3 degrees C-4.9 degrees C higher than the hydrogenated 1-acyl-glycerophosphoethanolamine at each pH investigated. At pH 9.0 and, more pronounced, at pH 10.5 we find a reduction of the transition temperature for both these substances, whereas their transition temperature is nearly unchanged at pH 4.0 and 7.2. Our results clearly show that the ether-bonding in the lysoderivative of plasmalogen is responsible for the closer packing compared to the 1-acyl-glycerophosphoethanolamine.

Alkenylhydrolase: a microsomal enzyme activity in rat brain.

Rat brain microsomes have the capacity to liberate radioactive free aldehydes from 1-[1-14C]alk-1'-enyl-sn-glycero-3-phosphoethanolamine (lysoplasmalogen). Glycerophosphoethanolamine was found using 1-alk-1'-enyl-sn-glycero-3-phospho-[3H]ethanolamine. The ratio of both products released by lysoplasmalogenase action was 1:1. Another enzymic activity could be demonstrated, which hydrolyzes lysoplasmalogen at the hydrophilic part of the molecule, a lysophospholipid phosphodiesterase. Thus, 1-[1-14C]alk-1'-enylglycerol was detected as well as [3H]ethanolamine, again in a molar ratio, from the respective labeled substrates. This enzyme possesses nearly the same affinity toward the substrate as lysoplasmalogenase. Whereas the lysophospholipid phosphodiesterase is totally inhibited in the presence of NaF or EDTA, lysoplasmalogenase activity is not affected by these reagents. 1-[1-14C]Alk-1'-enylglycerol acts also as substrate for lysoplasmalogenase, which liberates radioactive aldehydes at the same rate as from lysoplasmalogen. Because the apparent Km and Vmax values are nearly identical for both substrates, the enzyme activities are inhibited in the same way, and the pH optimum is about 7.2 in both cases, it is concluded that both substrates were attacked by the same enzyme. The enzyme does not differentiate between a substrate substituted at the sn-3 position of glycerol and one that is not. It requires only a free OH group at the sn-2 position. Phosphoethanolamine phosphatase activity was also determined under our experimental conditions.

On the phospholipid metabolism of glial cell primary cultures. II. Metabolism of 1-alkyl-glycerophosphoethanolamine during time course.

Primary cultures were prepared from newborn rat brain. After 16-18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1-[1-3H] alkyl-sn-glycero-3-phosphoethanolamine (1-alkyl-GPE), for 1-20 h. Five main products were formed: 1-alkyl-2-acyl-GPE; 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC); 1-alkenyl-2-acyl-GPE (ethanolamine plasmalogen); 1-alkenyl-2-acyl-GPC (choline plasmalogen); and 1-alkyl-glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmalogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl, 27.5% alkyl-acyl-, and 46.0% alkenyl-acyl-compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1-alkyl-glycerol was a minor reaction.

Lysoplasmalogenase--a microsomal enzyme from rat brain.

An enzymic activity of rat brain that liberates radioactive free aldehydes from 1-[1-14C]alk-1'-enyl-sn-glycero-3-phosphoethanolamine (lyso-plasmalogen) is described. It was present mainly in microsomal fractions (crude) of brains of rats of different ages. The highest specific enzyme activity was found in 21-day-old animals. The formation of free aldehyde was dependent on the amount of enzyme protein as well as the amount of substrate added, and was linear to the incubation time up to 60 min. The pH optimum was between 7.1 and 7.3. Bivalent cations (Mg2+, Ca2+) and detergents inhibited the reaction. However, the same cell fractions as well as extracts of acetone-dried powder of brain from young or old rats possessed no enzyme activity for liberating the aldehyde from the acylated substrates: 1-[1-14C]alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen) or plasmalogen of ox corpus callosum.

Phospholipid metabolism of glial cell primary cultures, IV. Metabolism of 1-alkenyl-sn-glycero-3-phosphoethanolamine between 1 and 20 hours incubation.

Primary cell cultures prepared from newborn rat brain were incubated on the 16th or 17th day with the substrate 1-([1-3H]-1-alkenyl)-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) for 1-20 h. The internalization of the substrate into the cells depended on the incubation time as well as on the amount of substrate. At any given time the acylation reaction to 2-acyl-1-alkenyl-sn-glycero-3-phosphoethanolamine (plasmalogen) was the most important event amounting to nearly 50-60% of the total radioactivity incorporated. Unchanged substrate was found in only small amounts within the cells. During incubation, the formation of 2-acyl-1-alkenyl-sn-glycero-3-phosphocholine (choline plasmalogen) increased, reaching saturation after 6 h with nearly 40% of the total radioactivity within the cells. These results were compared with those previously obtained with the substrate 1-([1-3H]alkyl)-sn-glycero-3-phosphoethanolamine under the same conditions. The acylation of this substrate as well as its conversion to the choline-containing analogue had been observed. Furthermore plasmalogen formation was also determined as a slow enzyme reaction. Both series of experiments showed a high acylation rate of 1-alkenylglycerophosphoethanolamine and a slow desaturation rate of the 1-alkyl compound. Thus, the following pathway of plasmalogen formation is proposed: 1-alkyl-sn-glycero-3-phosphoethanolamine leads to 1-alkenyl-sn-glycero-3-phosphoethanolamine leads to 2-acyl-1-alkenyl-sn-glycero-3-phosphoethanolamine.

Studies on the properties of phospholipase A1 in liver lysosomes of chloroquine-treated rats.

The influence of chloroquine on phospholipase A1 (acid pH optimum) activity was studied in subcellular fractions of rat liver after intraperitoneal application of the drug for 9, 12 and 21 days. In comparison with other cell fractions lysosomes of treated rats contained the highest enzyme activity with a pH optimum of 4.0. The activity of phospholipase A1 in lysosomes showed a direct relationship to the number of days of chloroquine treatment. The effects of the incubation time, Ca2+, Hg2+, enzyme and substrate concentration on phospholipase A1 activity were studied.

Liberation of free aldehyde from 1-(1-alkenyl)-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) by rat liver microsomes.

We found an enzyme in the microsomal fraction of 21-day-old-rat liver, which liberates a free aldehyde from 1-(1-alkenyl)-sn-glycero-3-phosphoethanolamine (lysoplasmalogen) and which has an activity of about 42 mU/mg protein under the conditions described. Kinetic data are presented. The pH optimum is found around pH 7.1. SH-blocking reagents, as well as deoxycholate, act as strong inhibitors, while Mg2 and Ca2 also inhibit the reaction to some extent. The enzymic activity is specific with respect to the monoradylphospholipid, since the acylated compound 2-acyl-1-(1-alkenyl)-sn-glycero-3-phosphoethanolamine does not serve as substrate. The ether linkage of 1-alkyl-sn-glycero-3-phosphoethanolamine is not hydrolyzed either under these conditions. A similar enzyme activity in liver has only been described for choline-containing lysoplasmalogen.

Studies on the hydrolysis of 1-alkyl-sn-glycero-3-phosphoethanolamine in subcellular fractions of rat brain.

The formation of 1-alkylglycerol from 1-alkyl-sn-glycero-3-phosphoethanolamine in different cell fractions of rat brain is reported. The substrates used were labelled either with 14C or 3H in the alkyl residue or with 14C in the alkyl and 3H in the ethanolamine residue. The examination of the lipid- and water-soluble cleavage products showed that both ethanolamine and phosphoethanolamine are liberated from the substrate in the microsomal fraction of 14-day-old rat brain. The latter product is rapidly hydrolyzed. In comparison with other cell fractions, the microsomes contained the highest enzyme activities, which exhibited a pH optimum of 7.1--7.5. SH-group reagents are inhibitors, whereas diisopropylfluorophosphate has no effect. As the animals age, these enzyme activities decrease in brain homogenates.