Incorporation Of Radioactivity Research Articles

RNA Modification and Digestive Tract Tumors: A Review.

Gastrointestinal tumors, including colorectal and liver cancer, are among the most prevalent and lethal solid tumors. These malignancies are characterized by worsening prognoses and increasing incidence rates. Traditional therapeutic approaches often prove ineffective. Recent advancements in high-throughput sequencing and sophisticated RNA modification detection technologies have uncovered numerous RNA chemical alterations significantly associated with the pathogenesis of various diseases, notably cancer. These discoveries have opened new avenues for therapeutic intervention. This article delves into epigenetic modifications, with a particular emphasis on RNA alterations such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), 1-methyladenosine (m1A), 7-methylguanosine (m7G), and N4-acetylcysteine (ac4C). It examines the functions and mechanisms of action of regulatory entities known as "Writers," "Readers," and "Erasers" to these modifications. Additionally, it outlines various methodologies for detecting these RNA modifications. Conventional techniques include radioactive isotope incorporation, two-dimensional thin-layer chromatography (2D-TLC), mass spectrometry, and immunological detection methods. Specialized methods such as bisulfite sequencing and reverse transcription stops are also discussed. Furthermore, the article underscores the significance of these modifications in the development, progression, and therapeutic targeting of gastrointestinal tumors, including esophageal, gastric, colorectal, liver, and pancreatic cancers. This exploration provides foundational insights for enhancing diagnostic accuracy, treatment efficacy, and prognostic assessment in gastrointestinal oncology.

Immunomodulatory properties of splenocytes treated with chlorpromazine in experimental aggression

Aggressive behavior is considered to be as one of the central symptoms of many neuropsychiatric disorders. It is a serious medical and biological problem associated both with high frequency and severity of manifestations and lack of selective correction means. The purpose of this study was to evaluate the functional phenotype of immune cells treated with chlorpromazine in aggressive animals in vitro, as well as the effect of transplantation of these cells on syngeneic aggressive recipient’s immune cells functional activity. Aggressive behavior was formed in active mice as a result of repeated experience of victories in agonistic interactions with animals with a submissive partner. Further, aggressive animals were isolated into individual cells and used as donors and recipients of immune cells. Immune cells were obtained under sterile conditions from suspension of spleen cells precultured with chlorpromazine. The level of spontaneous and mitogen-induced cell proliferation was assessed using a standard method of radioactive label incorporation. The quantitative content of cytokines in samples of treated with chlorpromazine cell cultures supernatant was determined by enzyme immunoassay using appropriate test systems. Further aggressive syngeneic recipients were intravenously injected with splenocytes precultured with chlorpromazine. The control group of animals was injected with splenocytes precultured under similar conditions, without the chlorpromazine addition. The recipients were assessed for the spleen cells proliferative activity and the intensity of humoral and cellular immune response using standard methods, by the number of antibody-forming spleen cells and severity of a delayed-type hypersensitivity reaction in response to T-dependent antigen introduction. It was found that treatment with chlorpromazine in vitro suppressed mitogen-stimulated proliferation of splenocytes in aggressive mice, without changing of spontaneous proliferation. It was also accompanied by some cytokines production decrease: IL-6, IL-2 and IFNã. When studying the humoral and cellular immune response intensity in aggressive recipients after transplantation of donor’s syngeneic splenocytes treated with chlorpromazine, a decrease in the intensity of humoral immune response was recorded. Transplantation of donor’s splenocytes treated with chlorpromazine was also accompanied by a decrease in spontaneous and mitogen-stimulated proliferation of splenocytes from aggressive recipients. Thus, the obtained data indicate the inhibitory effect of chlorpromazine on immune cell’s functional activity in aggressive mice, as well as the positive immunomodulatory effect of chlorpromazine-treated immune cells transplantation at aggressive behavior in experimental animals.

Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays.

ALPK1 is an atypical protein kinase that is activated during bacterial infection by ADP-heptose and phosphorylates TIFA to activate a cell signaling pathway. In contrast, specific mutations in ALPK1 allow it to also be activated by endogenous human nucleotide sugars such as UDP-mannose, leading to the phosphorylation of TIFA in the absence of infection. This protocol describes a quantitative, cell-free phosphorylation assay that can directly measure the catalytic activity of wildtype and disease-causing ALPK1 in the presence of different nucleotide sugars. In this method, overexpressed ALPK1 is first immunoprecipitated from the extracts of ALPK1 knockout HEK-Blue cells transfected with plasmids encoding either FLAG-tagged wildtype or mutant ALPK1, and then subjected to a radioactive phosphorylation assay in which the phosphorylation of purified GST-tagged TIFA by ALPK1 is quantified by measuring the incorporation of radioactivity derived from radiolabeled ATP. Key features • Quantitative measurement of protein kinase activity of wildtype and mutant ALPK1 in the presence or absence of different nucleotide sugars such as ADP-heptose and UDP-mannose. • Cell-free experimental setup overcoming the challenge of distinguishing constitutive activity and activation by endogenous mammalian metabolites in cell-based assays. • Requires approximately 50 µg of cell extract protein/reaction, allowing up to 150 assays to be performed from an extract prepared from a single 15 cm dish of transfected cells.

FASCIA Method in the Assessment of Lymphocyte Mitogen Responses in the Laboratory Diagnostics of Primary Immunodeficiencies

Lymphocyte responses to mitogens constitute a key part of the diagnostics of combined immunodeficiency (CID). Currently, mostly radioactive thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution methods are used. Flow-cytometric assay for specific cell-mediated immune-response in activated whole blood (FASCIA) has been put forth as an easy-to-perform option for the measurement of lymphocyte responses with the advantage of recognizing different lymphocyte subtypes and avoiding the use of radioactive reagents. Our aim was to analyze retrospectively the usefulness of FASCIA in the diagnostics of CID. We included all lymphocyte stimulation tests done with FASCIA in HUSLAB (Helsinki, Finland) between February 2015 and September 2018 in our analysis. The cohort was divided into two groups according to the patients’ final diagnoses: CID (n = 30) or non-CID (n = 159). We evaluated the stimulation responses with a combined FASCIA score (the average of all mitogen responses). The FASCIA score was significantly lower among the CID group compared to the other patients (p = 0.002), and in the ROC analysis, the AUC was 0.75 (p < 0.001) for the FASCIA score. When the three mitogens were analyzed separately, phytohemagglutinin (PHA) was best in separating patients with CID from non-CID (in the ROC analysis AUC 0.71, p = 0.001). Immunosuppressive medication affected the FASCIA result significantly and needs to be considered when evaluating the results. In conclusion, FASCIA can reliably detect the CID patients in the absence of immunosuppressive medication. It emerges as a method with many benefits compared to tests requiring radioactive reagents or the complicated CFSE staining.

Título en español.

Este valioso sistema enzimático puede tener muchas aplicaciones para trabajar en los más diversos campos científicos. Sería también, un sistema muy conveniente para futuros estudios sobre la biosíntesis de los esteroides en las plantas. Por otra parte, la incorporación de radiactividad dentro de esteroides sapogénicos podría simplificar la producción de aquellos productos farmacéuticos radiactivos derivados de éstos.

Effect of original anticonvulsant ortho-fluoro-benzonal on immune cells functional properties at chronic ethanol intoxication

IntroductionGABAa-receptors proved to be the molecular targets of ethanol on immune and nervous cells, potentiating alcohol influence. ortho-Fluoro-benzonal is known to be a circular urea derivative and an artificial ligand of GABA/BD-receptor and thus a potential candidate drug for alchoholism treatment.ObjectivesWe have shown the alcohol motivation decrease under ortho-fluoro-benzonal influence in experiment. The investigation of molecular mechanisms and functional targets of this substance is an important step in understanding of molecular pathogenesis and approaches to managing alcohol addiction.MethodsSplenocytes from male (CBAxC57Bl/6) F1 mice in a state of alcohol dependence owing to 6-month 10% ethanol exposure were aseptically obtained and cultured in presence of GABA, ortho-fluoro-benzonal and mitogens (LPS or concanavalin A). Proliferative activity of immune cells in vitro was estimated by means of radioactive 3H-thymidine incorporation.Results The intact animals’ splenocytes revealed increased spontaneous proliferation, increased T-mitogen stimulated and decreased B-mitogen stimulated proliferation in the presence of ortho-fluoro-benzonal. The immune cells from alcoholized animals, demonstrating increased spontaneous proliferative activity and weaken susceptibility to the mitogens, showed normal response patterns, except B-mitogen response case, under ortho-fluoro-benzonal inluence. Addition of GABA into the cultures didn’t cancel most positive effects of ortho-fluoro-benzonal inluence, proving existence of their GABAaR-independent pathways, mediated by other barbiturate receptors in addition to GABAaR-dependent ones.ConclusionsImmunomodulating properties of artificial GABA receptor ligand, ortho-fluoro-benzonal, in vitro has been shown. The compound may correct immune cells dysregulation caused by chronic ethanol exposure, so the original anticonvulsant has promise in the treatment of alcoholism.DisclosureThe authors have not supplied a conflict-of-interest statement.

PROBLEMATIC ASPECTS, COMPLICATIONS, MISCONCEPTIONS AND DEBATABLE ISSUES OF IODINE PROPHYLAXIS IN RADIATION EVENTS (REVIEW).

to consider and review the essence of emergency iodine prophylaxis during radiation events, physiological aspects of iodine metabolism in the body, properties of individual iodine prophylaxis agents that are recommended, and to justify the rationality of using some of them along with absurdity/inadmissibility of others; substantiate the creation of a unified preventive information strategy regarding the event in order to reduce anxiety and other negative psychological consequences among the affected population. The review was performed by searching the abstract and scientometric databases and printed publications. In the event of serious radiation events at nuclear power plants and industry facilities, radioactive iodine is highly likely to enter the environment. With the threat of radioactive iodine incorporation or with its incorporation that has already begun, it is absolutely necessary to carry out the emergency iodine prophylaxis. Such prevention should be carried out with stable iodine preparations such as potassium iodide or potassium iodate in special pharmaceutical formulas. Dosing of drugs in age and population groups should be carried out by specialists in radiation medicine and radiation safety in accordance with internationally recognized guidelines. The use of iodinecontaining food additives, iodine solution for external use and Lugol's solution is categorically unacceptable due to complete ineffectiveness, impracticality of implementation, and sometimes due to the threat of serious harm to health. Clear preparedness plans for possible radiation accidents and incidents, as well as successfullyimplemented appropriate preventive measures, including emergency iodine prophylaxis, are crucial for the effective and successful response to such events. Emergency iodine prophylaxis during radiation events should be carried out exclusively under the guidance of specialists in radiation medicine and radiation safety using special pharmaceutical formulas of potassium iodide or potassium iodate in doses recognized by the international scientific community. Other means of emergency iodine prophylaxis, including «handicraft»/home preparations, are absolutely unacceptable. Implementation of this protective measure should be accompanied by a coordinated information campaign in order to minimize purely radiation risks and to preserve the psychological well-being of the population.

Estimation of radiation-induced health hazards from a \u201cdirty bomb\u201d attack with radiocesium under different assault and rescue conditions

In the case of a terrorist attack by a “dirty bomb”, blast injuries, external irradiation and the incorporation of radioactivity are to be expected. Departing from information about the radiological attack scenario with cesium-137 in the U.S. National Scenario Planning Guide, we estimated the radiological doses absorbed. Similar calculations were performed for a smaller plume size and a detonation in a subway. For conditions as described in the U.S. scenario, the committed effective dose amounted to a maximum of 848 mSv, even for very unfavorable conditions. Red bone marrow equivalent doses are insufficient to induce acute radiation sickness (ARS). In the case of a smaller plume size, the ARS threshold may be exceeded in some cases. In a subway bombing, doses are much higher and the occurrence of ARS should be expected. The health hazards from a dirty bomb attack will depend on the location and the explosive device. The derived Haddon matrix indicates that preparing for such an event includes education of all the medical staff about radiation effects, the time lines of radiation damages and the treatment priorities. Further determinants of the outcome include rapid evacuation even from difficult locations, the availability of a specific triage tool to rapidly identify victims at risk for ARS, the availability of an antidote stockpile and dedicated hospital beds to treat seriously irradiated victims.

Osmotrophy of dissolved organic carbon by coccolithophores in darkness.

Summary The evolutionary and ecological story of coccolithophores poses questions about their heterotrophy, surviving darkness after the end‐Cretaceous asteroid impact as well as survival in the deep ocean twilight zone. Uptake of dissolved organic carbon might be an alternative nutritional strategy for supply of energy and carbon molecules.Using long‐term batch culture experiments, we examined coccolithophore growth and maintenance on organic compounds in darkness. Radiolabelled experiments were performed to study the uptake kinetics. Pulse–chase experiments were used to examine the uptake into unassimilated, exchangeable pools vs assimilated, nonexchangeable pools.We found that coccolithophores were able to survive and maintain their metabolism for up to 30 d in darkness, accomplishing about one cell division. The concentration dependence for uptake was similar to the concentration dependence for growth in Cruciplacolithus neohelis, suggesting that it was taking up carbon compounds and immediately incorporating them into biomass. We recorded net incorporation of radioactivity into the particulate inorganic fraction.We conclude that osmotrophy provides nutritional flexibility and supports long‐term survival in light intensities well below threshold for photosynthesis. The incorporation of dissolved organic matter into particulate inorganic carbon, raises fundamental questions about the role of the alkalinity pump and the alkalinity balance in the sea.

Diagnosing Wilson's Disease under the sword of Damocles.

Antos et al. [7] have reported a case of suspected uniparental disomy leading to an initial erroneous diagnosis of Wilson's Disease on the basis of genetic testing. They discuss the usefulness of the 64Cu radioactive copper incorporation test as an often-overlooked diagnostic aid. Wilson's Disease is difficult to diagnose because of its rarity, diverse clinical presentations, and the absence of a single fail-safe diagnostic test. The identification of mutations in the ATP7B gene has been an invaluable aid in the diagnosis, but genetic testing alone is not infallible, and should not be used as the sole diagnostic test in arriving at a diagnosis of Wilson's Disease. The diagnosis of Wilson's Disease must be based on a combination of findings that includes clinical history, clinical examination, and diagnostic testing. Genetic testing alone is insufficient.

Difficulties with diagnosing Wilson's disease by genetic testing alone

Objective: Diagnosis of Wilson's disease (WD) is based on the presence of clinical symptoms, laboratory (serum ceruloplasmin, serum copper, urinary copper excretion) and genetic findings. Diagnostic criteria of WD are summarized in Leipzig scoring. Radioactive copper incorporation is used when standard diagnostic criteria are insufficient.

Uptake of Soil-Derived Carbon into Plants: Implications for Disposal of Nuclear Waste.

Radiocarbon (14C) is potentially significant in terms of release from deep geological disposal of radioactive waste and incorporation into the biosphere. In this study we investigated the transfer of soil-derived C into two plant species by using a novel approach, where the uptake of soil-derived C into newly cultivated plants was studied on 8000-year leftover peat in order to distinguish between soil-derived and atmospheric C. Two-pool isotope mixing model was used to reveal the fraction of soil C in plants. Our results indicated that although the majority of plant C was obtained from atmosphere by photosynthesis, a significant portion (up to 3-5%) of C in plant roots was derived from old soil. We found that uptake of soil C into roots was more pronounced in ectomycorrhizal Scots pine than in endomycorrhizal reed canary grass, but nonetheless, both species showed soil-derived C uptake in their roots. Although plenty of soil-derived C was available in canopy air for reassimilation by photosynthesis, no trace of soil-derived C was detected in aboveground parts, possibly due to the open canopy. The results suggest that the potential for contamination with 14C is higher for roots than for leaves.

Apolipoprotein C-II Mimetic Peptide Promotes the Plasma Clearance of Triglyceride-Rich Lipid Emulsion and the Incorporation of Fatty Acids into Peripheral Tissues of Mice.

Aim Plasma apolipoprotein C-II (apoC-II) activates lipoprotein lipase (LPL) and thus lowers plasma triglycerides (TG). We previously reported that a human apoC-II mimetic peptide (C-II-a) decreased plasma TG in apoC-II mutant mice, as well as in apoE-knockout mice. Because it is unknown what tissues take up free fatty acids (FFAs) released from TG after C-II-a peptide administration, we investigated in mice TG plasma clearance and tissue incorporation, using 3H-triolein as a tracer, with and without C-II-a treatment. Methods and Results Intralipid® fat emulsion was labeled with 3H-triolein and then mixed with or without C-II-a. Addition of the peptide did not alter mean particle size of the lipid emulsion particles (298 nm) but accelerated their plasma clearance. After intravenous injection into C57BL/6N mice, the plasma half-life of the 3H-triolein for control and C-II-a treated emulsions was 18.3 ± 2.2 min and 14.8 ± 0.1 min, respectively. In apoC-II mutant mice, the plasma half-life of 3H-triolein for injected control and C-II-a treated emulsions was 30.1 ± 0.1 min and 14.8 ± 0.1 min, respectively. C57BL/6N and apoC-II mutant mice at 120 minutes after the injection showed increased tissue incorporation of radioactivity in white adipose tissue when C-II-a treated emulsion was used. Higher radiolabeled uptake of lipids from C-II-a treated emulsion was also observed in the skeletal muscle of C57BL/6N mice only. In case of apoC-II mutant mice, decreased uptake of radioactive lipids was observed in the liver and kidney after addition of C-II-a to the lipid emulsion. Conclusions C-II-a peptide promotes the plasma clearance of TG-rich lipid emulsions in wild type and apoC-II mutant mice and promotes the incorporation of fatty acids from TG in the lipid emulsions into specific peripheral tissues.

A non-radioactive in vitro CaMKII activity assay using HPLC-MS

IntroductionCalcium/Calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase that phosphorylates and regulates activity of many substrates in various tissues. Traditional CaMKII activity assays rely on incorporation of radioactivity onto a CaMKII substrate by utilizing γ-32P ATP, which has a short half-life and can pose health risks to the researchers. MethodsAn 8-minute HPLC-MS method was developed to measure a CaMKII-specific peptide substrate autocamtide-2 (AC-2) and its phosphorylated form, phosphoautocamtide-2 (PAC-2). Degradation of AC-2 and PAC-2 in solutions and how to stabilize them were studied. The method was validated according to FDA guidelines for bioassays, and applied to determine CaMKII activity in a C2C12 cell lysate and IC50 of KN-93, a known CaMKII inhibitor. ResultsSimple acidification with formic acid prevented AC-2 and PAC-2 from undergoing rapid degradation in the CaMKII assay mixture and in diluted water solutions. LLOQ of the HPLC-MS method was 0.26 μM and 0.12 μM for quantification of AC-2 and PAC-2, respectively. Precision was within 15% and accuracy was within 100 ± 15%. Using the developed method, IC50 of KN-93 was measured to be 399 ± 66 nM, which was compatible to reported values. ConclusionsA validated HPLC-MS method provides precise and accurate determination of AC-2 and PAC-2. This method enabled enzyme activity assay and inhibitor IC50 determination for CaMKII without radioactive labelled reagents.

Accuracy of the radioactive copper incorporation test in the diagnosis of Wilson disease.

In Wilson disease (WD), copper accumulates in the liver and other tissues because of mutations in the ATP7B copper transporter gene. Early and effective anticopper treatment is crucial. However, routine diagnostic methods based on clinical findings, copper metabolism tests, liver biopsies and DNA analyses do not always provide a conclusive diagnosis. The aim was to evaluate radioactive copper incorporation as a diagnostic test. We included cases with a diagnosis of WD supported by radiocopper testing and later, when available, confirmed by DNA analysis. Incorporation of 64 Cu was measured at 2, 24 and 48hours following intravenous injection. Diagnostic accuracy (area under the receiver operating characteristic curve [AUC]), sensitivity, specificity and predictive value were assessed for 24hours/2hours and 48hours/2hours 64 Cu ratios and compared with serum measurements of ceruloplasmin, copper, non-ceruloplasmin-bound copper and urinary 24-hours copper excretion. Patients having two pathogenic ATP7B mutations (homozygotes/compound heterozygotes) (n=74) had significantly lower 24hours/2hours and 48hours/2hours 64 Cu ratios than heterozygote controls (n=21) (mean 0.14 and 0.12 vs 0.49 and 0.63, respectively; both P<.001). Of note, 24hours/2hours and 48hours/2hours 64 Cu ratios had excellent diagnostic accuracy, with AUCs approaching 1, and only 24-hours urinary copper excretion displayed similar positive features. Other copper metabolism tests studied had lower accuracy, specificity and sensitivity. The radioactive copper test had excellent diagnostic accuracy and may be useful in the evaluation of new therapies aimed at restoring ATP7B function.

The environmental neurotoxin β-N-methylamino-l-alanine (l-BMAA) is deposited into birds' eggs

The neurotoxic amino acid β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disorders. BMAA is also a known developmental neurotoxin and research indicates that the sources of human and wildlife exposure may be more diverse than previously anticipated. The aim of the present study was therefore to examine whether BMAA can be transferred into birds’ eggs. Egg laying quail were dosed with 14C-labeled BMAA. The distribution of radioactivity in the birds and their laid eggs was then examined at different time points by autoradiography and phosphoimaging analysis. To evaluate the metabolic stability of the BMAA molecule, the distribution of 14C-methyl- and 14C-carboxyl-labeled BMAA were compared. The results revealed a pronounced incorporation of radioactivity in the eggs, predominantly in the yolk but also in the albumen. Imaging analysis showed that the concentrations of radioactivity in the liver decreased about seven times between the 24h and the 72h time points, while the concentrations in egg yolk remained largely unchanged. At 72h the egg yolk contained about five times the concentration of radioactivity in the liver. Both BMAA preparations gave rise to similar distribution pattern in the bird tissues and in the eggs, indicating metabolic stability of the labeled groups. The demonstrated deposition into eggs warrants studies of BMAAs effects on bird development. Moreover, birds’ eggs may be a source of human BMAA exposure, provided that the laying birds are exposed to BMAA via their diet.

A new non-radioactive deoxyhypusine synthase assay adaptable to high throughput screening.

Deoxyhypusine synthase (DHS) catalyzes the post-translational modification of eukaryotic translation factor 5A (eIF5A) by the polyamine, spermidine, that converts one specific lysine residue to deoxyhypusine [N ε -4-aminobutyl(lysine)], which is subsequently hydroxylated to hypusine [N ε -4-amino-2-hydroxybutyl(lysine)]. Hypusine synthesis represents the most critical function of polyamine. As eIF5A has been implicated in various human diseases, identification of specific inhibitors of hypusine modification is of vital importance. DHS catalyzes a complex reaction that occurs in two stages, first, the NAD-dependent cleavage of spermidine to form an enzyme-butylimine intermediate and enzyme-bound NADH, and second, the transfer of the butylimine moiety from the enzyme intermediate to the eIF5A precursor and subsequent reduction of the eIF5A-butylimine intermediate by enzyme-bound NADH to form deoxyhypusine [N ε -4-aminobutyl(lysine)]. Our data demonstrate that there is a measurable release of enzyme-bound NADH in the absence of eIF5A precursor and that the DHS activity can be determined by coupling the first phase reaction with the NADH-Glo assay in which the generation of luminescence is dependent on NADH derived from the DHS partial reaction. The conventional DHS assay that measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A precursor in the complete reaction cannot be readily adapted for high throughput screening (HTS). In contrast, the non-radioactive DHS/NADH-Glo coupled assay is highly specific, sensitive and reproducible and could be configured for HTS of small molecule libraries for the identification of new inhibitors of DHS. Furthermore, the coupled assay provides new insights into the dynamics of the DHS reaction especially regarding the fate of NADH.

Conjunction of mitochondrial targeting nutrients for the management of heart dysfunctions associated with type 2 diabetes

Backgrounds and aims The heart is known to be susceptible to oxidative damage from high ambient oxidant levels, hypercoagulation and hypofibrinolysis associated with diabetes. It is also likely that poly(ADP-ribose)polymerase-1 (PARP-1) activation plays a crucial role in the pathogenesis of heart dysfunctions induced by diabetes. The study was performed to evaluate cardioprotective efficacy of a combined supplementation of mitochondria-specific antioxidants [acetyl-L-carnitine (ALC) and alpha-lipoic acid (ALA)] and nicotinamide (NAm), as a multiple-action drug in prevention of metabolic and haemostatic system's abnormalities induced by diabetes. Material and methods Neonatal diabetes was induced by STZ (80mg/kg b. w., i.p.) on the 2nd day of male Wistar rats' life. Rats were treated for 2 wks with or without combination of ALC, ALA and NAm (respectively 100, 50 and 100mg/kg b.w., i.p.) after 12 wks of STZ-induced diabetes. PARP-1 activity in heart was measured by the incorporation of radioactivity from NAD+ (labeled in the adenine moiety) into nuclei proteins. The metabolic and hemostatic system's parameters were also assessed. Results After 14 wks diabetic animals had not lost weight compared with control and treatment did not affect it, P P P P P Conclusion The findings suggest that combined supplement of naturally occurring biologically active compounds such as ALC, ALA and NAm offer a non-toxic, well tolerated and rational option for heart dysfunctions associated with type 2 diabetes improving metabolic and haemostatic system's alterations.

Phospholipid changes in Rhinella arenarum embryos under different acclimation conditions to copper

We report phospholipid changes in Rhinella arenarum embryos after applying three acclimation protocols to copper between 40 and 420ngL−1. The lower and higher acclimation treatments resulted in embryos' enhanced resistance to this metal. Phospholipid remodeling activity, evident through arachidonic acid radioactivity incorporation increase in phosphatidylcholine (PC) and sphingomyelin (SPH) fractions, was registered in embryos acclimated to the intermediate exposure condition. Concomitantly, a decrease in phosphatidic acid fraction (PA) was registered in the higher acclimation condition. PC/PE radioactivity ratio increased both for medium and high acclimation conditions from 0.493 in control embryos to 1.378 and 1.032 respectively. Phospholipid changes could be relevant for changes in membrane features associated with low level exposures to copper, preparing the embryo for a higher resistance to this metal. The increased resistance to copper could also be associated with both an increase in metallothioneins concentration, as registered with HPLC in all the acclimation conditions, and an increase in the copper bound to the third fraction of metallothioneins separated by this method. Our results point out that even very low level exposure to copper results in phospholipid metabolism changes that could be relevant for the acclimation phenomena.

A Technique for the Measurement of in vitro Phospholipid Synthesis via Radioactive Labeling

This is an assay designed to examine the radioactive phosphorous incorporation when the molecule is being synthesized, which means that only de novo synthesized phospholipids can be detected. Thus, with this technique it is possible to detect in vitro phospholipid synthesis under different required experimental conditions respect to controls (Guido and Caputto, 1990; Ferrero et al., 2014). There are different types of lipids. Among them we can find phospholipids, which contain glycerol esterified with two fatty acyl chains and a phosphate group that can also be bound to an organic molecule that acts as “hydrophilic head”, as shown in Figure 1 for the case of phosphatidylcholine. This structure confers amphipathic properties to lipid molecules that allow them to form lipid bilayers, making phospholipids the main components of biological membranes. Figure 1. Representation of phospholipid structure. Extracted from: http://bio1151.nicerweb.com/Locked/media/ch05/phospholipid.html