Radioactive Labeling Techniques Research Articles

Metabolic Study of Tetra-PEG-Based Hydrogel after Pelvic Implantation in Rats.

In vivo metabolism of polyethylene glycol (PEG) hydrogels has rarely been studied. In this study, we prepared a chemically crosslinked hydrogel formulation using 14C-labeled tetra-armed poly (ethylene glycol) succinimidyl succinate (Tetra-PEG-SS) and 3H-labeled crosslinking agent for implantation into the pelvis of Sprague-Dawley (SD) rats. This radioactive labeling technique was used to investigate the radioactivity excretion rates in of feces and urine, the blood exposure time curve, and the radioactivity recovery rate in each tissue over time. We showed that the primary excretion route of the hydrogel was via urine (3H: about 86.4%, 14C: about 90.0%), with fewer portion through feces (3H: about 6.922%, 14C: about 8.16%). The hydrogel metabolites exhibited the highest distribution in the kidney, followed by the jejunal contents; The 3H and 14C radioactivity exposures in the remaining tissues were low. We also showed that the 3H and 14C radioactivity recovery rates in the blood were usually low (<0.10% g−1 at 12 h after implantation), even though, in theory, the hydrogel could be absorbed into the blood through the adjacent tissues. By using a combination of HPLC-MS/MS and offline radioactivity counting method, we established that the tetra-PEG-based hydrogel was mainly metabolized to lower-order PEG polymers and other low-molecular-weight substances in vivo.

In vivo analysis of protein translation activity in sea urchin eggs and embryos.

Protein synthesis is a major regulatory step of gene expression in different physiological processes including development. Translation of proteins in sea urchin is stimulated upon fertilization and is necessary for cell cycle progression and development. Translational control is exerted through multifactorial mechanisms, including mRNA recruitment into polysomes and increased rates of translational activity. In this chapter, we review the methods used in sea urchin eggs and embryos to analyze translation activity in vivo both from perspectives of the proteins and of the mRNAs. First, we describe methods to quantify or visualize newly synthesized proteins with radioactive and non-radioactive labeling techniques. Next we present the polysome isolation and profiling on sucrose gradients, allowing the identification of translated mRNAs. Finally, we outline a procedure to follow the translation of a reporter luciferase protein from an mRNA microinjected into the egg.

Oral nanotherapeutics: Redox nanoparticles attenuate ultraviolet B radiation-induced skin inflammatory disorders in Kud:Hr- hairless mice.

The active participation of an anti-inflammatory drug in the biological pathways of inflammation is crucial for the achievement of beneficial and therapeutic effects. This study demonstrated the development of redox nanoparticles that can circulate in the blood at significantly high levels, thus increasing their efficacy as an oral treatment against the deleterious effects of reactive oxygen species (ROS) in an invivo inflammatory skin model. To confirm the blood bioavailability of the nanoparticles, mice were injected with the nanoparticles solution (RNPN) via oral gavage. Using electron spin resonance and radioactive labeling techniques, the blood circulation of the redox polymer that forms the nanoparticles was confirmed 24h after oral administration. This contrasted with its low molecular weight counterpart (NH2-TEMPO), which peaked 15min post injection and was found to be cleared rapidly within minutes after the peak. We then tested its efficacy in the inflammatory skin model. Kud:Hr-hairless mice were irradiated with UVB (302nm) to induce skin damage and inflammation. Throughout the entire period of UVB irradiation, RNPN was administered to mice by free drinking. NH2-TEMPO was used as the control. The results showed that oral supplementation of RNPN significantly improved the therapeutic effects of the core nitroxide radical compared with its low molecular weight counterpart. Furthermore, RNPN significantly reduced UVB-induced skin aging, epidermal thickening, edema, erythema, skin lesions, and various pathological skin inflammatory disorders invivo. From the obtained data, we concluded that the use of long-circulating redox nanoparticles (RNPN) provided an effective treatment against the damaging effects of excessive ROS in the body.

Drivers of Phosphorus Uptake by Barley Following Secondary Resource Application.

Minable rock phosphate is a finite resource. Replacing mineral phosphorus (P) fertilizer with P-rich secondary resources is one way to manage P more efficiently, but the importance of physicochemical and microbial soil processes induced by secondary resources for plant P uptake is still poorly understood. Using radioactive-labeling techniques, the fertilization effects of dairy manure, fish sludge, meat bone meal, and wood ash were studied as P uptake by barley after 44 days and compared with those of water-soluble mineral P (MinP) and an unfertilized control (NoP) in a pot experiment with an agricultural soil containing little available P at two soil pH levels, approximately pH 5.3 (unlimed soil) and pH 6.2 (limed soil). In a parallel incubation experiment, the effects of the secondary resources on physicochemical and microbial soil processes were studied. The results showed that the relative agronomic efficiency compared with MinP decreased in the order: manure ≥fish sludge ≥wood ash ≥meat bone meal. The solubility of inorganic P in secondary resources was the main driver for P uptake by barley (Hordeum vulgare). The effects of secondary resources on physicochemical and microbial soil processes were of little overall importance. Application of organic carbon with manure resulted in microbial P immobilization and decreased uptake by barley of P derived from the soil. On both soils, P uptake by barley was best explained by a positive linear relationship with the H2O + NaHCO3-soluble inorganic P fraction in fertilizers or by a linear negative relationship with the HCl-soluble inorganic P fraction in fertilizers.

Brassinosteroid-induced de novo protein synthesis in Zea mays under salinity and bioinformatic approach for identification of heat shock proteins

Using radioactive labeling technique it was analyzed proteins profiles of maize plants after treatment (12 h) with salt and brassinosteroids (BRs). Among separated proteins 66 and 67 proteins were observed specifically upregulated by salt treatment or BRs under salinity, respectively. Brassinazole—inhibitor of BRs biosynthesis—lowered BR-induced accumulation of proteins that confirm their BR-mediated synthesis. Data on 200 heat shock proteins (HSPs) from Arabidopsis thaliana and 42 HSPs from Zea mays have been used for polypeptide identification with bioinformatic approach. Protein bands separated on SDS-PAGE were compared with gene expression profile of HSPs from Genevestigator database in response to salinity and hormone treatment. In result, eight correlative matches were found between levels of polypeptide accumulation and activation of HSPs genes’ expression. It was suggested involvement of BRs in regulation of synthesis of mitochondrial chaperones under salinity.

Biodynamic modelling of the bioaccumulation of trace metals (Ag, As and Zn) by an infaunal estuarine invertebrate, the clam Scrobicularia plana

Biodynamic modelling was used to investigate the uptake and accumulation of three trace metals (Ag, As, Zn) by the deposit feeding estuarine bivalve mollusc Scrobicularia plana. Radioactive labelling techniques were used to quantify the rates of trace metal uptake (and subsequent elimination) from water and sediment diet. The uptake rate constant from solution (±SE) was greatest for Ag (3.954±0.375lg−1d−1) followed by As (0.807±0.129lg−1d−1) and Zn (0.103±0.016lg−1d−1). Assimilation efficiencies from ingested sediment were 40.2±1.3% (Ag), 31.7±1.0% (Zn) and 25.3±0.9% (As). Efflux rate constants after exposure to metals in the solution or sediment fell in the range of 0.014–0.060d−1. By incorporating these physiological parameters into biodynamic models, our results showed that dissolved metal is the predominant source of accumulated Ag, As and Zn in S. plana, accounting for 66–99%, 50–97% and 52–98% of total accumulation of Ag, As and Zn, respectively, under different field exposure conditions. In general, model-predicted steady state concentrations of Ag, As and Zn matched well with those observed in clams collected in SW England estuaries. Our findings highlight the potential of biodynamic modelling to predict Ag, As and Zn accumulation in S. plana, taking into account specific dissolved and sediment concentrations of the metals at a particular field site, together with local water and sediment geochemistries.

A fluorescent method to determine vitamin K-dependent gamma-glutamyl carboxylase activity

The gamma (γ)-glutamyl carboxylase is a key enzyme in vitamin K-dependent carboxylation of proteins involved in hemostasis and inflammation. It is an endoplasmic enzyme posttranslationally converting glutamic acid residues into γ-carboxyglutamic acid residues in proteins. The activity of tissue derived γ-glutamyl carboxylase is commonly assayed by incorporation of H14CO3- into synthetic peptides and subsequent quantification using liquid scintillation counting. We present a nonradioactive assay using a fluorescein isothiocyanate-labeled short peptide that can be readily detected in its unmodified and γ-glutamyl carboxylated form by reversed-phase HPLC. This method offers a convenient alternative to the established radioactive labeling techniques.

In vitro study of the biological interface of Bio‐Oss: implications of the experimental setup

To systematically investigate the biological interface of Bio-Oss by analysing dissolution-precipitation behaviour and osteogenic responses using in vitro experimental systems. Different concentrations (1-100mg/ml) of Bio-Oss were incubated in cell culture medium for 24h before elemental concentrations for calcium, phosphorus and silicon in the medium were analysed with inductive coupled plasma-optical emission spectroscopy. Radioactive calcium-45 isotope labelling technique was used to study possible precipitation of calcium on the Bio-Oss particle. Biological interface of Bio-Oss was studied in osteogenic experiments using mineralization medium and three different sources of cells (primary mouse bone marrow stromal cells, primary rat calvarial cells and MC3T3-E1 mouse pre-osteoblast cell line). Cells were fixed and stained with Toulidine blue, von Kossa or Alizarin Red staining for confirmation of extracellular matrix mineralization. Elemental analysis of the cell culture medium demonstrated a significant decrease of calcium and phosphorus and a dose-dependent release of silicon to the medium after incubation with Bio-Oss. A significant decrease of calcium and phosphorus in the medium occurred even at low concentrations of Bio-Oss. Uptake of calcium on the Bio-Oss particle was confirmed with radioactive calcium-45 isotope labelling technique. In osteogenic experiments with Bio-Oss (<1mg/ml), matrix mineralization around the Bio-Oss particles were demonstrated in all three cell types with von Kossa and Alizarin Red staining. Dissolution-precipitation reactions occur at the surface of Bio-Oss, and osteogenic responses are seen at the biological interface. The concentration of Bio-Oss is a key factor for the experimental in vitro results, and may also have implications for the clinic.

Deletion of synapsins I and II genes alters the size of vesicular pools and rabphilin phosphorylation

Previous studies established that genetic deletion of synapsins, synaptic vesicle-associated phosphoproteins that regulate neurotransmitter release, decreases the number of synaptic vesicles in nerve terminals. To investigate whether these changes affect the release properties of the remaining synaptic vesicles, we used a radioactive labeling technique to measure release independently of the total number of synaptic vesicles. 3H-glutamate and 14C-γ-amino-butyric-acid (GABA) release from isolated nerve terminals prepared from the neocortex of synapsins I and II double knock-out mice (DKO) was assayed and compared to wild-type preparations. Hyperosmotic shock-evoked 3H-glutamate was reduced by 20 ± 3% from DKO nerve terminals and potassium depolarization-evoked glutamate release was also decreased by 28 ± 2%. Surprisingly, sucrose or potassium depolarization-evoked release of 14C-GABA was increased by 32 ± 4% and 29 ± 5%, respectively. The basal efflux of both 3H-glutamate and 14C-GABA increased by 17 ± 2% and 12 ± 2% from DKO nerve terminals. As lack of synapsins I and II, major phosphoproteins of synaptic vesicles, may lead to deregulation of phosphorylation events, we compared phosphorylation state of another synaptic vesicle protein, rabphilin. In DKO nerve terminals, membrane-associated rabphilin level was reduced by ∼0.28-fold, its phosphorylation at 234serine was increased by ∼1.61-fold whereas cytosolic rabphilin levels showed both more dramatic reduction in abundance, ∼16.5-fold, and increase in phosphorylation, ∼4.8-fold. Collectively, these data suggest that deletion of major synapsin isoforms leads to (1) deregulation of basal neurotransmission causing “leaky” basal release, (2) changes in either the size or mobilization of releasable or reserve pools, and (3) a decrease in rabphilin abundance accompanied by an increase in basal phosphorylation of the remaining rabphilin.

Non-fusion expression in Escherichia coli: Single-step purification of recombinant human annexin A5 for detection of apoptosis

Recombinant human annexin A5 (rh-annexin A5) was originally used to detect early stages of apoptosis in vitro. With the development of radioactive labeling and imaging techniques, annexin A5 labeled with radioactive markers can play a more important role in monitoring apoptotic cells in vivo. To obtain highly pure rh-annexin A5 with an easy and inexpensive purification approach, we constructed a pJLA503-annexin A5 expression plasmid, which could overexpress human annexin A5 in a soluble form in Escherichia coli. Then a novel purification method based on Ca 2+-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of rh-annexin A5 was increased to 98%. To confirm the PS affinity of rh-annexin A5 produced by this purification protocol, a simple and reliable lipid membrane model was prepared and used in the binding test. As a probe to detect apoptosis, the fluorescein isothiocyanate-labeled rh-annexin A5 was incubated with apoptotic cells. The results showed that the labeled rh-annexin A5 possessed high affinity for PS molecule and was able to indicate different apoptotic states.

Penetration and loading of human serum albumin in porous silicon layers with different pore sizes and thicknesses

Human serum albumin was adsorbed into porous silicon layers with thickness up to 3 μm and with different mean pore radius in the range 4.5–10 nm. The adsorbed amount of protein was quantified by I 125 radioactive labeling techniques and ellipsometry. The results show that albumin penetrated into the pores when the mean pore radius was larger than 5.5 nm, but could not totally occupy the available surface area when the layer thickness was larger than 1 μm. Loading of albumin both into porous layers and onto plane silicon as a function of albumin concentration was also investigated. These measurements show that loading of protein increased with protein concentration at least up to 10 mg/ml for porous silicon and up to 1 mg/ml for plane silicon. The maximum deposition into the type of porous layers used here was 28 μg/cm 2, compared to 0.36 μg/cm 2 for plane silicon.

Quercetin sensitizes cells in a tumour-like low pH environment to hyperthermia

Quercetin has been shown to act as a hyperthermia sensitizer by inhibiting the synthesis of heat shock protein 70 (HSP70) in a variety of tumour cell lines. It is most effective under conditions of low pH. This study was designed to test the hypothesis that quercetin suppresses thermotolerance development in cells adapted to growth at low pH and renders them as responsive as acutely acidified cells to hyperthermia-induced cytotoxicity. Chinese hamster ovarian carcinoma cells (OvCa) were exposed to 42°C hyperthermia and/or quercetin (50-200 mm) at their growth pH of either 7.3 or 6.7 or after acute acidification from 7.3 to 6.7. Thermotolerance development was measured by colony survival. HSP70 synthesis and total protein synthesis were measured by radioactive precursor pulse labelling techniques. Quercetin, in a concentration-dependent manner, reduced the rate of total protein synthesis and increased cytotoxicity equally after acute acidification to pH 6.7 or growth at pH 6.7 at 37°C, and to a greater extent than it did in cells at pH 7.3. At 42°C, 100 mm quercetin inhibited total protein synthesis, HSP70 synthesis and thermotolerance development to a similar extent in cells grown at pH 6.7 or acutely acidified to pH 6.7. In contrast, quercetin reduced but did not completely inhibit HSP70 synthesis and thermotolerance development in cells grown and heated at pH 7.3. These results support the hypothesis that quercetin can specifically reduce thermotolerance development in tumour cells adapted to growth at pH e 6.7 so that they respond similarly to acutely acidified cells. Since many tumours are adapted to growth at low pH and may resist a wide variety of therapeutic modalities, inhibition of thermotolerance expression by quercetin may not only enhance the response to hyperthermia but the response to commonly used therapies such as chemotherapy and radiation.

Radioactive Labeling Study and FTIR Measurements of Methanol Adsorption and Oxidation on Fuel Cell Catalysts

AbstractThe adsorption and the electrooxidation of methanol‐derived surface CO on fuel‐cell grade platinum and platinum‐ruthenium alloy nanoparticle catalysts were studied using a combination of electrochemical measurements and radioactive labeling (thin foil radioactive labeling technique) and in situ infrared reflectance spectroscopy. The kinetics of formation of surface and bulk products coming from methanol and surface/bulk exchange processes were found to be significantly different on Pt compared to Pt/Ru. The observed difference in the kinetics of CO adsorption underlines the electronic effect in the surface catalytic activity due to modification of the electronic structure of surface platinum atoms by ruthenium. Correlation between FTIR and radiolabeling measurements indicated that the adsorption of CO from methanol is a very slow process at low potential but that the coverage by CO poison is greater at higher potential. Then, the results obtained from both analytical techniques (in situ IR reflectance spectroscopy and radioactive labeling method) displayed very good agreement and allowed us to validate them for the investigation of the methanol oxidation mechanism.

Prognostic significance of extensive microsatellite instability in sporadic clinicopathological stage C colorectal cancer.

Colorectal cancers exhibiting microsatellite instability (MSI) appear to have unique biological behaviour. This study analyses the association between extensive MSI (MSI-H), clinicopathological features and survival in an unselected group of patients with sporadic Australian Clinico-Pathological Stage (ACPS) C (tumour node metastasis stage III) colorectal cancer. Some 255 patients who underwent resection for sporadic ACPS C colorectal cancer between 1986 and 1992 were studied. No patient had received chemotherapy. Minimum follow-up for all patients was 5 years. Archival normal and tumour DNA was extracted and amplified by polymerase chain reaction using a radioactive labelling technique. MSI-H was defined as instability in 40 per cent or more of seven markers. Twenty-one patients showed MSI-H. No association was found between MSI and age or sex. Tumours exhibiting MSI-H were more commonly right sided (P<0.00001), larger (P = 0.002) and more likely to be high grade (P = 0.049). After adjustment for age, sex and other pathological variables, patients whose cancers exhibited MSI-H had improved survival (P = 0.015). Recognition of MSI-H in sporadic ACPS C tumours identifies a subset of cancers with improved prognosis. Such stratification should be considered in trials of adjuvant therapy and may be relevant to therapeutic decision making.

Nonradioactive labeling of large DNA fragments for genome walking, RFLP and northern blot analysis.

In this report, we present a simple nonradioactive labeling procedure for DNA fragments of high specific labeling density that can be used for a variety of applications. The protocol is based on the universal mono-functional platinum reagent for chemical digoxigenin (DIG) labeling of nucleic acids. The labeling protocol was optimized for large DNA templates as complete bacterial artificial chromosomes (BAC). Variations of incubation time and temperature improved the labeling density such that about 30% of the nucleotides were DIG-modified within 30 min. Furthermore, the refined procedure generates in a single-tube reaction and without prior digestion-labeled DNA fragments of 0.5-4.0 kb from a 130-kb template. Hybridization experiments were performed on Southern and northern blots and allowed the detection of single copy genes in 2.5 micrograms genomic DNA from Arabidopsis thaliana, which has a haploid genome size of 0.13 pg (ca. 120 Mb) and medium expressed transcripts from 0.8 microgram poly(A)+ RNA, respectively. The extremely high specific labeling density, the stability and the universal application of the probe generated with the platinum reagent makes this method a useful alternative to classical radioactive nuclei acids labeling techniques.

Elution of Adsorbed Fibrinogen from a Silica Surface by Anionic Surfactants. 2. Effect of the Hydrocarbon Chain Lengths

The chain length effect of 8, 10, and 14 carbon homologous sodium alkyl sulfate surfactants on the elution of fibrinogen molecules adsorbed onto a silica surface has been studied. Previously, it has been shown (Zembala et al. Langmuir 1998, 14, 2167) that the release mechanism of fibrinogen by sodium dodecyl sulfate (SDS) is the result of a competition between the elution and anchoring of the adsorbed proteins. All of the removal kinetics, followed by a radioactive labeling technique, could be described by a single-exponential decay function. The apparent release rate constant k and the amount of released proteins increase with the surfactant concentration cs and attain plateau values for cs close to the critical micelle concentration (cmc). By means of two-step elution experiments (first, low-concentration surfactant injection, followed by the injection of surfactants above cmc), it was shown that all surfactants were able to anchor fibrinogen molecules onto the solid surface and that surfactants with a smaller chain length possess a higher ability to anchor proteins than surfactants with longer chains. On the contrary, for a given surfactant concentration, elution increases with the chain length.

Flow injection with chemical reaction interface–isotope ratio mass spectrometry: an alternative to off-line combustion for detecting low levels of enriched 13C in mass balance studies

We have evaluated the potential of flow injection chemical reaction interface isotope-ratio mass spectrometry to replace radioactive labeling techniques in material balance studies. A sample is flow injected and transmitted through a desolvation system followed by combustion to form 13CO 2 with a microwave-powered chemical reaction interface. We can detect trace amounts of a 13C-labeled drug (3′-azido-3′-deoxythymidine, AZT) in urine or feces. Our ability to quantify less than 100 ng/mL of excess 13C (∼1 μg/mL of 13C-labeled AZT) from a sample equivalent to 10 μL of urine is superior to previous detection limits for 13C in urine that use off-line combustion methods. Parallel studies using 14C-labeled AZT showed that our stable isotope method provides comparable percent excretion data for urine and feces. These results support previous findings that mass balance studies could be carried out with isotope-ratio mass spectrometer, here using doses as low as 1–2 mg/kg.

Adsorption Kinetics and Ligand-Binding Properties of Thiol-Modified Double-Stranded DNA on a Gold Surface

The development of DNA-based biosensors requires efficient immobilization of DNA probes on the sensor surfaces with optimum coverage and orientation. In this study, we have prepared thin films of a 718 base pair 5‘-thiol-labeled double-stranded DNA (dsDNA) on a gold surface by chemisorption and determined the quantity, surface coverage, and adsorption kinetics of dsDNA using cyclic voltammetry (CV), quartz crystal resonator (QCR), and radioactive labeling techniques. The adsorption and desorption rate constants of the 5‘-thiol-labeled dsDNA on a gold surface are estimated to be (1.9 ± 0.2) × 103 M-1 s-1 and (1.3 ± 1.1) × 10-3 s-1, respectively. The equilibrium constant is (1.5 ± 1.3) × 106 M-1 and the fractional coverage is >75% for the DNA concentration range of 2−10 μM. The ligand-binding properties of the immobilized dsDNA were investigated using doxorubicin, a DNA-intercalating anthracyclic antibiotic, as the probing molecule. The binding of doxorubicin to the immobilized dsDNA was examined by CV to v...

Enzymatic fluorescence and biotin labeling of primers for PCR sequencing.

AbstractThe emergence of cycle sequencing (1) as a powerful alternative to conventional isothermal methods has facilitated the manipulations involved in sequencing protocols in general, and is of value for the sequence analysis of PCR products, in particular. Using radioactive labeling techniques, it has generally been accepted that 5′-terminal phosphorylation of the primer prior to the sequencing reaction (2) is the most reliable approach to obtaining sequence information. However, the combination of nonisotopic detection with cycle sequencing offers several alternatives. The incorporation of the fluorescent label into the nascent DNA has been approached from several angles. At present, one may consider three sources of fluorescence (Fig. 1), irrespective of the principle governing the design of the sequencing hardware: 1. Label attached at the 5′-terminus of the primer (3). 2. Label incorporated as an appropriately chemically modified dideoxy-terminating nucleotide (4). 3. Internal labeling of the growing chain using F-dATP during the elongation reaction (5). Source of label during fluorescent DNA sequencing. The fluorescent tag is indicated schematically by a star. KeywordsSequencing ProtocolTerminal TransferaseUnincorporated NucleotideElongation ReactionObtain Sequence InformationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A simple technique for surface labeling of solids with222Rn and its short-lived decay products

A simple radioactive labeling technique is described which is based on a recoil injection of atoms of222Rn and its decay products from a226Ra source into thin surface layers of solids.