A procedure for the synthesis of p-fluorosulfonyl[14C]-benzoyl-5′-adenosine with [14C] in the benzoyl moiety

Carboxy-p-fluorosulfonyl[¹⁴C]benzoyl-5′-adenosine has been synthesized with the radiolabel ultimately derived from carboxy-p-amino[¹⁴C]benzoic acid by a synthetic route employing four reaction steps. Starting with 1 mmol of p-amino[¹⁴C]benzoic acid, p-fluorosulfonyl[¹⁴C]benzoyl-5′-adenosine is obtained with an overall yield of 25–30%.     

Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-¹⁴C]glucosamine or l-[U-¹⁴C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.     

Assay of Δ1-piperideine-2-carboxylate and synthesis of l-[14C]pipecolate from dl-[14C]pipecolate

Four assay methods were tested for the measurement of Δ¹-piperideine-2-carboxylate, a proposed alicyclic ketimino acid intermediate in the pathway of lysine metabolism to l-pipecolate, and the product of d-amino acid oxidase on d-pipecolate. The method using Δ¹-piperideine-2-carboxylate reductase from Pseudomonas putida was found to be most sensitive and specific. Measurement of Δ¹-piperideine-2-carboxylate by reduction with NaBH₄ and ninhydrin assay of the resultant pipecolate, by direct acidic ninhydrin assay, and by o-aminobenz-aldehyde assay were less desirable because of lower sensitivity and specificity. Two synthetic methods for preparing l-[¹⁴C]pipecolate from the racemic dl-[¹⁴C]pipecolate were investigated. Incubation of dl-[¹⁴C]pipecolate with a combination of d-amino acid oxidase and Δ¹-piperideine-2-carboxylate reductase or d-amino acid oxidase and NaBH₄ totally inverted the d-isomer to the l-isomer, with $\text{Δ}{}^1\text{-[}{}^{14}\text{C]piperideine-2-carboxylate}$ as an intermediate in each cycle of interconversion. No purification except desalting through a Dowex 50 (H⁺) column was necessary in order to recover l-[¹⁴C]pipecolate in pure form. The yield was 95–97% compared to <50% in the conventional method.     

Interaction of α-cyano[14C]cinnamate with the mitochondrial pyruvate translocator

The binding of α-cyanocinnamate to rat-heart mitochondrial membrane was investigated using α-cyano[¹⁴C]cinnamate. The binding was correlated to the inhibition of pyruvate transport. The results obtained demonstrate that both these functions reach saturation at the same titre of the inhibitor. Quantitative parameters of α-cyano[¹⁴C]cinnamate binding have been determined. The binding can be prevented by pyruvate and other substrates of the carrier but not by acetate. Pyruvate decreases the affinity of α-cyanocinnamate binding, leaving the maximum number of binding unchanged. It is concluded that rat-heart mitochondria contain a specific site at which α-cyanocinnamate binds which is directly involved in the inhibition of pyruvate transport.     

Transport und Stoffwechsel von 2-[14C]Abscisinsäure in Wurzelsegmenten von Phaseolus coccineus L.

Summary Movement of 2-[¹⁴C]ABA through 1.5 cm and 5 cm long root segments of P. coccineus L. was acropetally polarised. The velocity of acropetal transport of [¹⁴C]ABA in 1.5 cm long segments was 4–5 mm·h^-1. Up to 11 h after the start of incubation [¹⁴C]ABA could be extracted unchanged. Beyond this time radioactivity became associated to an unidentified compound, which shows chromatographic qualities similar to those of Milborrow's Metabolite C (Biochemistry & Physiol. Plant Growth Substances, 1531, Runge Press Ottawa, 1968; and Chem. Comm. 966, 1969).

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Abkürzungen ABA Abscisinsäure - PPO 2,5-Diphenyloxazol     

Incorporation of label from [1-14C]ethanol into the glutamate–glutamine pools of rat brain in vivo

Glutamate and glutamine were isolated from the brains of rats given [1-(14)C]ethanol by three different routes of injection with and without carrier ethanol or acetate. Without exception, the specific radioactivity of brain glutamine was 3- to 5-fold that of brain glutamate. The route of injection had no consistent effect on the results. These results indicate that small amounts of ethanol are oxidized by rat brain in the compartment responsible for the so-called ;small metabolic pool' of brain glutamate.     

Dietary α-Linolenic Acid Increases the Biosynthesis of the Choline Glycerophospholipids from [14C]CDPcholine in Rat Liver and Kidney But Not in Brain

The effect of feeding rats for 30 days with diets containing high levels of linoleic acid (sunflower oil, SO) or -linolenic acid (perilla oil, PO) was studied in the liver, kidney and brain. The PO group showed a higher labeling of choline glycerophospholipids (CGP) in liver and kidney but no difference with the SO group in ethanolamine glycerophospholipids (EGP) labeling. The brain displayed the lowest incorporation of both precursors and no difference between the two diets. Analyses of brain CGP and EGP fatty acid compositions showed that in the PO group the ratio n-6/n-3 was lower than in the SO group, mainly as a consequence of lower levels of n-6 fatty acids. The mole % of docosahexaenoate (DHA) in these lipids was the same for both groups and only triacylglycerols (TAG) displayed a higher DHA. Therefore, at least in the brain, the magnitude of fatty acid changes observed in CGP and EGP for the PO group does not affect the uptake/incorporation of the precursors into phospholipids.     

Fate of [carbonyl-14C]methabenzthiazuron in an arid region soil — effect of organic amendment,soil disturbance and fumigation

[Carbonyl-¹⁴C] methabenzthiazuron (MBT) was applied to an arid region soil at a rate of 5mg kg⁻¹ soil to give a¹⁴C content of 2400 KB kg⁻¹ soil. After 15 weeks of incubation at 22°C and 50% of the maximum water holding capacity of the soil, 7.2% of the applied¹⁴C was mineralized to¹⁴CO₂. Where the soil was amended with wheat straw, total mineralization increased to 17.3%. Soil disturbance caused a significant increase while chloroform fumigation caused a significant decrease in the rate of¹⁴CO₂ production, both from amended and unamended soils. These results suggest that MBT is degraded mainly through microbial co-metabolism. Wheat straw amendment resulted in increased transformation of MBT into soil humus. In unamended soil, a major portion of¹⁴C was recovered in fulvic acid and in fractions extracted with organic solvents. Recovery of¹⁴C in non-extractable bound residues (humins) increased as incubation progressed and seemed to be derived from the fulvic acid fraction, which showed a concomitant decrease. More than 99% of the residual¹⁴C in unamended soil consisted of unaltered MBT; the remainder occurred as 1-methyl-1 (benzthiazolyl) urea. In amended soil, a relatively higher percentage of the extractable¹⁴C was found in the metabolite. Small amounts of three unidentified¹⁴C-labelled compounds were also observed. In amended soil, disturbance caused a decrease in extractable-¹⁴C whereas fumigation caused a significant increase, as compared to the untreated control. The effects were more pronounced when the soils were reated at an early stage of incubation. In general, soil disturbance increased the availability of MBT for further transformations while chloroform fumigation decreased the process.     

Differential effects of veratridine and calcium on the release of [3H]noradrenaline and [14C]α-aminoisobutyrate from rat brain cortex slices

The efflux of [³H]noradrenaline (NA) and of the non transmitter, non metabolizable, amino acid [¹⁴C]α-aminoisobutyrate (AIB), was followed simultaneously from superfused rat brain cortex thin slices, that had been preloaded with those substances. Short (2 min) “pulses” of increasing veratridine concentrations were applied at 10 min intervals. When calcium in the superfusion fluid was 1 mM, [³H]NA efflux increased progressively with pulses of 1, 3, 10 and 30 μM veratridine, but further increase to 100 μM resulted in a decrease of the induced ³H-efflux. Veratridine-enhanced [³H]NA efflux decreased considerably in 0.1 mM calcium and was virtually suppressed when no calcium was added to the superfusion fluid. In 1 mM calcium, the efflux of [¹⁴C] AIB was increased progressively by pulses of 10, 30 and 100 μM veratridine, but no increase in efflux was seen with 1 or 3 μM drug. In 0.1 mM, or without added calcium, the induced efflux of [¹⁴C]AIB was markedly increased. Similar findings were seen when a long (10 min) pulse of 10 μM veratridine was given. After such long pulses there was a rapid return of AIB efflux to pre-veratridine levels if calcium was 1 mM, but in the absence of added calcium, the return to baseline levels of both [³H]NA and, especially, that of [¹⁴C]AIB efflux, was greatly impaired. The veratridine enhanced efflux of both NA and AIB was entirely blocked by 1 μM tetrodotoxin.     

Development and endocrine regulation of mitochondrial cytochrome biosynthesis in the insect fat body: δ-[14C]aminolevulinic acid incorporation

The rate of incorporation of [¹⁴C]aminolevulinic acid (ALA) into cytochrome hemes was used to measure mitochondrial cytochrome synthesis in the fat body of adult male Blaberus discoidalis cockroaches. The hemes of cytochromes aa₃+b and c+c₁, were chemically separated to observe differential rates in their synthesis and regulation. [¹⁴C]ALA was linearly incorporated into cytochrome hemes for at least 8 h. No significant pool of endogenous ALA was detected relative to the amount of administered [¹⁴C]ALA. Peak cytochrome synthesis occurred 4 to 6 days after adult emergence. Endocrine disruption by corpora cardiaca-corpora allata extirpation or cervical ligation eliminated the 4-day developmentally related increase in the rate of cytochrome aa₃+b synthesis but had no effect on the production of cytochromes c+c₁. Injections of corpora cardiaca extracts into cervically ligated animals stimulated the rate of production of cytochromes aa₃+b by 2.5 times but did not affect cytochromes c+c₁. By comparison, juvenile hormone injections did not affect the rate of synthesis of either cytochrome fraction. These findings indicate that a neurohormone regulates the rate of synthesis of cytochromes a+b in insect fat body mitochondria.     

双链核糖核酸免疫化学研究 Ⅰ.双链多聚[I]:多聚[C]的抗原性

用国产多聚肌苷酸和多聚胞嘧啶核苷酸(简称多聚[I]:多聚[C])制备成双链核糖核酸特异性抗血清,用环状沉淀和~3H标记的番茄花叶病毒双链核糖核酸测得抗血清效价分别为1:128和1:6400。用免疫琼脂双扩散、对流免疫电泳和放射免疫测定可测定出多聚[I]:多聚[C]的最低浓度分别为391ng、12.2/ng和10pg/ml抗血清和酵母、TMV、PVX 的单链核糖核酸、小牛胸腺DNA不反应。试验证明用国产多聚[I]:多聚[C]制备的抗血清,可有效地用干双链核糖核酸的免疫化学鉴定。由于在免疫双扩散和对流免疫电泳中抗血清能和微量双链核糖核酸反应并产生可见沉淀线,从而有可能利用这两种简便灵敏的方法测定病毒的双链RNA,单链RNA病毒的RF型RNA,以及双链RNA真菌病毒的筛选。     

Human mycoses in Portugal [1960–1973]

A critical survey is made of human mycoses diagnosed in European Portugal and in the Portuguese Overseas Provinces. Dermatophyte infections and pityriasis versicolor are commom in the entire territory. The more frequently isolated dermatophytes in Continental Portugal wereTrichophyton violaceum, Microsporum canis, Trichophyton tonsurans andTrichophyton schoenleinii, in the scalp andTrichophyton rubrum, Trichophyton mentagrophytes, Trichophyton megninii andEpidermophyton floccosum in the other body sites. In the Overseas Provinces the species found in white people were very much the same, whereas in negroes mainlyMicrosporum audouinii andT. violaceum in Mozambique,M. audouinii in Angola, andTrichophyton soudanense, M. audouinii andT. rubrum in Guinea were identified.In Continental Portugal cases of candidiasis, mycetoma, aspergillosis, sporotrichosis and cryptococcosis were described in patients who had never been outside Europe; and tinea nigra, African histoplasmosis and South-American blastomycosis in individuals who live or lived in India, Africa and Brazil.The first case of North-American blastomycosis was reported in Mozambique.     

Truncated cystatin C in cerebrospiral fluid: Technical [corrected] artefact or biological process?

Cystatin C, a low molecular weight cysteine proteinase inhibitor present in human body fluids at physiological concentrations, is more expressed in cerebrospinal fluid (CSF) than in plasma. Mass spectrometric characterization showed that after 3 months of storage of human CSF at -20 degrees C, cystatin C was cleaved in the peptide bond between R8 and L9 and lost its eight N-termini amino acids, whereas this cleavage did not occur when stored at -80 degrees C. This truncation occurred in all CSF samples studied irrespective of the underlying neurological status, indicating a storage-related artefact rather than a physiological or pathological processing of the protein. These results stress the importance of optimal preanalytical storage conditions of any sample prior to proteomics studies.     

CaMKIIδC slows [Ca]i decline in cardiac myocytes by promoting Ca sparks

Acute activation of calcium/calmodulin-dependent protein kinase (CaMKII) in permeabilized phospholamban knockout (PLN-KO) mouse myocytes phosphorylates ryanodine receptors (RyRs) and activates spontaneous local sarcoplasmic reticulum (SR) Ca release events (Ca sparks) even at constant SR Ca load. To assess how CaMKII regulates SR Ca release in intact myocytes (independent of SR Ca content changes or PLN effects), we compared Ca sparks in PLN-KO versus mice, which also have transgenic cardiac overexpression of CaMKIIδC in the PLN-KO background (KO/TG). Compared with PLN-KO mice, these KO/TG cardiomyocytes exhibited 1), increased twitch Ca transient and fractional release (both by ～35%), but unaltered SR Ca load; 2), increased resting Ca spark frequency (300%) despite a lower diastolic [Ca]i, which also slowed twitch [Ca]i decline (suggesting CaMKII-dependent RyR Ca sensitization); 3), elevated Ca spark amplitude and rate of Ca release (which might indicate that more RyR channels participate in a single spark); 4), prolonged Ca spark rise time (which implies that CaMKII either delays RyR closure or prolongs the time when openings can occur); 5), more frequent repetitive sparks at single release sites. Analysis of repetitive sparks from individual Ca release sites indicates that CaMKII enhanced RyR Ca sensitivity, but did not change the time course of SR Ca refilling. These results demonstrate that there are dramatic CaMKII-mediated effects on RyR Ca release that occur via regulation of both RyR activation and termination processes.     

In Vivo Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists [11C]A-582941 and [11C]A-844606 in Mice and Conscious Monkeys

Background

The α7 nicotinic acetylcholine receptors (nAChRs) play an important role in the pathophysiology of neuropsychiatric diseases such as schizophrenia and Alzheimer''s disease. The goal of this study was to evaluate the two carbon-11-labeled α7 nAChR agonists [¹¹C]A-582941 and [¹¹C]A-844606 for their potential as novel positron emission tomography (PET) tracers.

Methodology/Principal Findings

The two tracers were synthesized by methylation of the corresponding desmethyl precursors using [¹¹C]methyl triflate. Effects of receptor blockade in mice were determined by coinjection of either tracer along with a carrier or an excess amount of a selective α7 nAChR agonist (SSR180711). Metabolic stability was investigated using radio-HPLC. Dynamic PET scans were performed in conscious monkeys with/without SSR180711-treatment. [¹¹C]A-582941 and [¹¹C]A-844606 showed high uptake in the mouse brain. Most radioactive compounds in the brain were detected as an unchanged form. However, regional selectivity and selective receptor blockade were not clearly observed for either compound in the mouse brain. On the other hand, the total distribution volume of [¹¹C]A-582941 and [¹¹C]A-844606 was high in the hippocampus and thalamus but low in the cerebellum in the conscious monkey brain, and reduced by pretreatment with SSR180711.

Conclusions/Significance

A nonhuman primate study suggests that [¹¹C]A-582941 and [¹¹C]A-844606 would be potential PET ligands for imaging α7 nAChRs in the human brain.     

Urinary cell cycle arrest biomarker [TIMP-2]·[IGFBP7] in patients with hepatorenal syndrome

Abstract

Background: Patients with hepatorenal syndrome carry a high short-term mortality. Early diagnosis and treatment are essential for patients’ outcome. Nevertheless diagnosis of HRS remains difficult. First-line therapy terlipressin is often associated with severe complications. Biomarkers become more on focus for an early diagnosis.

Objective: The aim of this study was to test the diagnostic accuracy of urinary [TIMP-2]·[IGFBP7] for HRS patients and prognostic value for therapy responding patients.

Material and methods: NephroCheck^® measures urinary concentrations of TIMP-2 and IGFBP-7, both indicating stress of renal cells and associated with induction of cell cycle arrest. 22 HRS patients and 30 patients with normal kidney function were included. [TIMP-2]·[IGFBP7] was measured using NephroCheck^®. HRS patients receiving terlipressin were also examined.

Results: [TIMP-2]·[IGFBP7] values did not differ significantly (1.3?±?2.09 vs. 1.03?±?1.03; p?=?0.55). Furthermore, there was no significant difference of [TIMP-2]·[IGFBP7] regarding response of terlipressin (1.32?±?2.39 vs. 0.81?±?1.05; p?=?0.56). Low [TIMP-2]·[IGFBP7] values were significantly associated with higher mortality (p?=?0.01).

Conclusions: The results of this study suggest that [TIMP-2]·[IGFBP7] is not suitable for diagnostic of HRS and prediction of therapy response, but there might be evidence for prognostic value of [TIMP-2]·[IGFBP7] in regard to mortality of liver cirrhosis patients.     

Use of [4,6-Di-14C]-5′-DMT-thymidine Phosphoramidite Reagent for the Radiolabeling of Synthetic Oligonucleotides

Abstract

A novel synthesis of 5′-radiolabeled oligonucleotides is described. The labeling is carried out by the phosphoramidite method with the aid of building block 1. The feasibility of the method is demonstrated by preparation of 5′-radiolabeled 3′-phosphorylated dodecathymidylate phosphorothioate.     

Decarboxylative metabolism of [1′-14C]indole-3-acetic acid by tomato pericarp discs during ripening

The rate of decarboxylation of [1′-¹⁴C]indole-3-acetic acid (IAA) infiltrated into tomato (Lycopersicon esculentum Mill.) pericarp discs was much more rapid in green than in breaker and pink tissues. Studies were carried out in order to determine whether the decarboxylative catabolism occurring in the green pericarp discs was associated with ripening or was a consequence of wound-induced peroxidase activity and/or ethylene production. After a 2-h lag, the decarboxylative capacity of the green pericarp discs increased exponentially during a 24-h incubation period. This increase was accompanied by increases in IAA-oxidase activity in cell-free preparations from the intercellular space and cut surface of the discs. Although higher IAA-oxidase activity was detected in extracts from the tissue residue, which comprises mainly intracellular peroxidases, this activity did not increase during the 24-h incubation period. Analysis of the cell-free preparations by isoelectric focusing revealed the major component in all samples was a highly anionic peroxidase (pI=3.5) the levels of which did not increase during incubation. However, the intercellular and cut-surface preparations contained additional anionic and cationic peroxidases which increased in parallel with the increases in both the IAA-oxidase activity of the preparations and the decarboxylative capacity of the green pericarp discs from which they were derived. Treatment of green discs with the ethylene-biosynthesis inhibitors aminooxyacetic acid and CoCl₂, inhibited the development of an enhanced capacity to decarboxylate [1′-¹⁴C]IAA but the inhibition was not counteracted by exogenous ethylene. Another ethylene-biosynthesis inhibitor, aminoethoxyvinyl glycine, also reduced ethylene levels but did not affect IAA decarboxylation, indicating that the decarboxylation was not a consequence of wound-induced ethylene production. The data obtained thus demonstrate that the enhanced capacity to decarboxylate [1′-¹⁴C]IAA that develops in green tomato pericarp discs following excision is not associated with ripening but instead is attributable to a wound-induced increase in anionic and cationic peroxidase activity in the intercellular fluid and at the cut surface of the excised tissues.