Macrocyclic complexes of scandium radionuclides as precursors for diagnostic and therapeutic radiopharmaceuticals

The aim of this study was to evaluate new ligands which can be applied for labeling biomolecules with scandium radionuclides. Two radionuclides of scandium, ⁴⁷Sc and ⁴⁴Sc, are perspective radioisotopes for radiotherapy and diagnostic imaging. ⁴⁷Sc decays with a half-life of 3.35 days and a maximum β⁻ energy of 600 keV and could be an alternative to carrier added ¹⁷⁷Lu radionuclide for targeted radionuclide therapy. Another scandium radionuclide ⁴⁴Sc (t_1/2 = 3.92 h) is an ideal β⁺ emitter for PET diagnosis. It can be obtained as a daughter of the long-lived ⁴⁴Ti (t_1/2 = 60.4 y) from ⁴⁴Ti/⁴⁴Sc generator. For complexation of scandium radionuclides macrocyclic ligands having a cavity size similar to Sc³⁺ ionic radius were selected: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7 triacetic acid (NOTA), 1,4,7-triazacyclodecane-1,4,7 triacetic acid and 1,4,7-triazacycloundecane triacetic acid, and analogs of NOTA with 10, 11 and 12 atoms of the carbon in the ring. Our results have shown that from the studied macrocyclic ligands studied DOTA is most efficient for binding scandium radionuclides ⁴⁴Sc and ⁴⁷Sc to biomolecules. The determined stability constant of Sc-DOTA complex logK = 27.0 is comparable with stability constants for Y³⁺ and heaviest lanthanides but is higher than those for In³⁺ and Ga³⁺. Also ⁴⁶Sc-DOTATATE conjugate exhibits high stability in-vitro studies. The ¹³C NMR studies have shown that Sc-DOTA like Lu-DOTA forms in solution complexes with eight-coordination geometry. The lipophilicity of Sc-DOTATATE is nearly identical to that of Lu-DOTATATE, which suggests similar receptor affinity of both radioconjugates.     

Tritium labelling of the amino acids of Sinapis alba is identical in darkness and far-red light

Earlier reports that irradiation for 3 hr with continuous far-red light stimulated the incorporation of deuterium from deuterium oxide into the free amino acids of the cotyledons of Sinapis alba have not been confirmed by studies using tritium oxide. Possible explanations of the discrepancy are discussed.     

Rapid mixing between old and new C pools in the canopy of mature forest trees

Stable C isotope signals in plant tissues became a key tool in explaining growth responses to the environment. The technique is based on the fundamental assumption that the isotopic composition of a given unit of tissue (e.g. a tree ring) reflects the specific C uptake conditions in the leaf at a given time. Beyond the methodological implications of any deviation from this assumption, it is of physiological interest whether new C is transferred directly from sources (a photosynthesizing leaf) to structural sinks (e.g. adjacent stem tissue), or inherently passes through existing (mobile) C pools, which may be of variable (older) age. Here, we explore the fate of (13)C-labelled photosynthates in the crowns of a 30-35 m tall, mixed forest using a canopy crane. In all nine study species labelled C reached woody tissue within 2-9 h after labelling. Four months later, very small signals were left in branch wood of Tilia suggesting that low mixing of new, labelled C with old C had taken place. In contrast, signals in Fagus and Quercus had increased, indicating more intense mixing. This species-specific mixing of new with old C pools is likely to mask year- or season-specific linkages between tree ring formation and climate and has considerable implications for climate reconstruction using stable isotopes as proxies for past climatic conditions.     

Cadmium stress induces changes in the lipid composition and biosynthesis in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.) leaves

The effects of cadmium (Cd) stress on lipid composition and biosynthesis were investigated in young leaves of ten-day-old tomato seedlings (Lycopersicon esculentum Mill. cv. Ibiza F1). Cd was found to be mainly accumulated in roots, but a severe inhibition of biomass production occurred in leaves, even at its low concentration (1.0 μM). Seven days after Cd treatment, the membrane lipids were extracted and separated on silica-gel thin layer chromatography (TLC). Fatty acid methyl esters were analyzed by FID-GC on a capillary column. Our results showed that Cd stress decreased the quantities of all lipids classes (phospholipids, galactolipids and neutral lipids). Likewise, there was also a decline in the levels of tri-unsaturated fatty acids, such as linolenic (C18:3) and hexadecatrienoic (C16:3) acids. The linolenic acid (C18:3) decreased in monogalactosyldiacylglycerol (MGDG) and all phospholipids, while hexadecatrienoinic acid (C16:3) declined mainly in MGDG. Moreover, Cd at high concentrations (25.0 and 50.0 μM) significantly enhanced the levels of lipid peroxides. Radiolabelling experiments were carried out by laying down microdroplets of [1-¹⁴C]acetate–a major precursor of lipid biosynthesis–on attached leaves of the control and Cd-treated plants. After incubation for 1, 2, 12 and 24 h, the leaves were harvested and lipids extracted and analysed. Cd stress was found to decrease the incorporation of [1-¹⁴C]acetate in total lipids. The biosynthesis of total lipids was altered with 25.0 and 50.0 μM Cd. The decline in the incorporation of [1-¹⁴C]acetate due to Cd stress was observed in all lipid classes. There was also a substantial decline in the incorporation of [1-¹⁴C]acetate in tri-unsaturated fatty acids. The results indicate that Cd treatment induces an oxidative stress by inhibiting the chloroplastic and extrachloroplastic lipid-biosynthesis pathways as well as lipid peroxidation.     

Rubidium marking of Anopheles mosquitoes detectable by field-capable X-ray spectrometry

We present a mosquito marking technique suitable for mark-release-recapture that can be used with a hand-held, portable X-ray fluorescence (XRF) spectrometer, which is practical for field measurements. Third instar Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) and Anopheles stephensi Liston larvae were cultured to pupation in water containing rubidium (Rb) Cl at concentrations up to 1000 p.p.m. Rb. Anopheles gambiae larvae survived to adulthood at concentrations as high as 1000 p.p.m. Rb but suffered pupal mortality and reduced adult longevity at high concentrations. We were able to culture An. stephensi at Rb concentrations as high as 300 p.p.m. The presence of Rb in adults was evaluated using a portable XRF analyser, and we were able to reliably detect Rb above background levels in 10-day-old females and 4-day-old males at concentrations causing minimal pupal or adult mortality. We observed that Rb marking was not permanent, and the concentration declined significantly as adults aged. The low cost of labelling with RbCl and the field portability of the spectrometer provide a useful means for labelling mosquitoes via breeding sites or in the laboratory for mark-release-recapture experiments.     

Mechanisms of gonadal morphogenesis are not conserved between chick and mouse

To understand mechanisms of sex determination, it is important to know the lineage relationships of cells comprising the gonads. For example, in mice, the Y-linked gene Sry triggers differentiation of Sertoli cells from a cell population originating in the coelomic epithelium overlying the nascent gonad that also gives rise to uncharacterised interstitial cells. In contrast, little is known about origins of somatic cell types in the chick testis, where there is no Sry gene and sex determination depends on a ZZ male/ZW female mechanism. To investigate this, we performed fate mapping experiments in ovo, labelling at indifferent stages the coelomic epithelium by electroporation with a lacZ reporter gene and the underlying nephrogenous (or mesonephric) mesenchyme with chemical dyes. After sex differentiation, LacZ-positive cells were exclusively outside testis cords and were 3betaHSD-negative, indicating that the coelomic epithelium contributes only to non-steroidogenic interstitial cells. However, we detected dye-labelled cells both inside and outside the cords. The former were AMH-positive while some of the latter were 3betaHSD-positive, showing that nephrogenous mesenchyme contributes to both Sertoli cells and steroidogenic cells. This is the first demonstration via lineage analysis that steroidogenic cells originate from nephrogenous mesenchyme, but the revelation that Sertoli cells have different origins between chick and mouse suggests that, during evolution, mechanisms of gonad morphogenesis may diverge alongside those of sex determination.     

Altered molecular specificity of surfactant phosphatidycholine synthesis in patients with acute respiratory distress syndrome

Background

Acute respiratory distress syndrome (ARDS) is a life-threatening critical illness, characterised by qualitative and quantitative surfactant compositional changes associated with premature airway collapse, gas-exchange abnormalities and acute hypoxic respiratory failure. The underlying mechanisms for this dysregulation in surfactant metabolisms are not fully explored. Lack of therapeutic benefits from clinical trials, highlight the importance of detailed in-vivo analysis and characterisation of ARDS patients according to patterns of surfactant synthesis and metabolism.

Methods

Ten patients with moderate to severe ARDS were recruited. Most (90%) suffered from pneumonia. They had an infusion of methyl-D₉-choline chloride and small volume bronchoalveolar lavage fluid (BALF) was obtained at 0,6,12,24,48,72 and 96 hours. Controls were healthy volunteers, who had BALF at 24 and 48 hours after methyl-D₉-choline infusion. Compositional analysis and enrichment patterns of stable isotope labelling of surfactant phosphatidylcholine (PC) was determined by electrospray ionisation mass spectrometry.

Results

BALF of patients with ARDS consisted of diminished total PC and fractional PC16:0/16:0 concentrations compared to healthy controls. Compositional analysis revealed, reductions in fractional compositions of saturated PC species with elevated levels of longer acyl chain unsaturated PC species. Molecular specificity of newly synthesised PC fraction showed time course variation, with lower PC16:0/16:0 composition at earlier time points, but achieved near equilibrium with endogenous composition at 48 hours after methyl-D₉-choline infusion. The enrichment of methyl-D₉-choline into surfactant total PC is nearly doubled in patients, with considerable variation between individuals.

Conclusions

This study demonstrate significant alterations in composition and kinetics of surfactant PC extracted from ARDS patients. This novel approach may facilitate biochemical phenotyping of ARDS patients according to surfactant synthesis and metabolism, enabling individualised treatment approaches for the management of ARDS patients in the future.     

Defense activation triggers differential expression of phospholipase-C (PLC) genes and elevated temperature induces phosphatidic acid (PA) accumulation in tomato

Recently, we provided the first genetic evidence for the requirement of tomato PLC4 and PLC6 genes in defense activation and disease resistance. The encoded enzymes were catalytically active as they were able to degrade phosphatidylinositol (PI), thereby producing diacylglycerol (DG). Here we report differential PLC gene expression following the initiation of defense signaling by the interaction between Cladosporium fulvum resistance (R) protein Cf-4 and its matching effector Avr4 in tomato hybrid seedlings that express both Cf-4 and Avr4. Furthermore, we observed that PLC3 and PLC6 gene expression is upregulated by elevated temperature in the control seedlings. This upregulation coincides with an increase in the levels of phosphatidic acid (PA) and a decrease in the levels of PI and phosphatidylinositol phosphate (PIP). The decrease in PI and PIP levels matches with the activation of PLC. In addition, the levels of the structural phospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) declined transiently during recovery after the exposure to elevated temperature., Further studies will be required to explain the mechanism causing the sustained accumulation of PA during recovery, combined with a reduction in the levels of structural phospholipids.