Effect of albumin on the kinetics of ascorbate oxidation

The fluorescence intensity of the fluorophore in dansyl piperidine-nitroxide is intramolecularly quenched by the nitroxyl fragment. Therefore, the oxidation of ascorbic acid by the fluorophore-nitroxide (FN) probe can be monitored by two independent methods: steady-state fluorescence and electron paramagnetic resonance. Bovine serum albumin (BSA) affects the rate of this reaction. The influence of BSA on the rate is attributed to the adsorption of both ascorbate and the probe to BSA. Adsorption of ascorbate to BSA is confirmed by NMR relaxation experiments. The spatial distribution of the molecules on the BSA surface changes the availability of ascorbate and FN to each other. The results also point out that, in the presence of BSA, the autoxidation of ascorbate is significantly slowed down. The effect is studied at different pH values and explained in terms of the electrostatic interaction between the ascorbate anion and the BSA molecule.     

Lead concentrates in ovarian follicle compromises pregnancy

Following absorption, lead can concentrate in bodily compartments where it disrupts cellular processes and can result in detrimental health consequences. The concentration and impact of lead within follicular fluid has not been characterized and we used inductively coupled plasma mass spectroscopy (ICP-MS) to determine lead levels in blood and follicular fluid from nine patients undergoing in vitro fertilization (IVF) treatment. Lead levels within follicular fluid were found to be significantly higher in non-pregnant patients compared to pregnant patients suggesting that elevated concentrations of the environmental toxicant lead adversely affect female reproduction.     

The reduction of a nitroxide spin label as a probe of human blood antioxidant properties

The kinetics of reduction of the radical R*, 5-dimethylaminonaphthalene-1-sulfonyl-4-amino-2,2,6,6-tetramethyl-1-piperidine-oxyl by blood and its components were studied using the EPR technique. The results demonstrate that R* is adsorbed to the outer surface of the membrane and does not penetrate into the erythrocytes. A series of control experiments in PBS demonstrate that ascorbate is the only natural reducing agent that reacts with R*. The observed first order rate of disappearance of the nitroxide radical k, is: k(blood) > k(eryth) > k(plasma) and k(blood) approximately = k(eryth) + k(plasma). The results demonstrate that: a. The erythrocytes catalyze the reduction of R* by ascorbate. b. The rate of reduction of the radical is high though it does not penetrate the cells. c. In human erythrocytes there is an efficient electron transfer route through the cell membrane. d. The study points out that R* is a suitable spin label for measuring the reduction kinetics and antioxidant capacity in blood as expressed by reduction by ascorbate.     

Neural responses to antigenic challenges and immunomodulatory factors

In studies designed to examine the effects of the immune system, recordings of multi-unit electrical activity (MUA) in the central nervous system were made in the preoptic area/anterior hypothalamus (POA/AH) and paraventricular nucleus (PVN) of rats, following sheep red blood cell (SRBC) immunization. Peak increases in POA/AH MUA were observed on the fifth day following SRBC sensitization, the day on which serum antibodies were first detected. A significant increase in paraventricular nucleus (PVN) MUA was also observed on the sixth day. These changes in POA/AH and PVN MUA were found to be associated with increased plasma corticosterone levels on day 8. Induction of a second immune response to SRBC evoked a POA/AH MUA increase of extended duration but reduced amplitude, while administration of the immunosuppressive drug, cyclophosphamide, prevented both antibody generation and any increases in POA/AH MUA. These data suggest that activation of the immune system may provide signals, in the form of chemical messengers, which are able to alter neural activity in some regions of the brain that are important in neuroendocrine regulatory mechanisms. Accordingly, intracerebroventricular injections of thymic humoral factor or alpha-interferon decreased POA/AH MUA, increased EEG synchronization, and decreased plasma levels of corticosterone. Histamine and interleukin 1 did not alter POA/AH MUA but decreased EEG synchronization and increased plasma levels of corticosterone.     

Effects of the Serotonin1A Agonist, 8-Hydroxy-2-(Di-n-Propylamino)tetralin on Neurochemical Responses to Stress

Abstract: The effects of intracerebroventricular administration of the 5-hydroxytryptamine (5-HT)_1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.1 pmol) on adrenocortical and neurochemical responses to stress were examined in conscious male rats. The following stress paradigms were used: acoustic stimulation (105 dB for 2 min); footshock (0.2 mA, five shocks over 5 min); conditioned fear (animals placed in a footshock chamber for 5 min, 24 h after footshock); restraint (5 min); intraperitoneal (i.p.) injection of recombinant human interleukin-1α (rHu-IL-1α, 20 µg/kg); and injection of cocaine hydrochloride (20 mg/kg, i.p.). As previously shown, 8-OH-DPAT was able to attenuate the adrenocortical response to acoustic stress, conditioned fear, rHu-IL-1α, and cocaine administration. Cocaine decreased 5-hydroxyindoleacetic acid (5-HIAA)/5-HT and dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratios and norepinephrine (NE) concentration in the prefrontal cortex, hypothalamus, and brainstem in all experiments, and 8-OH-DPAT reversed the changes in DOPAC/DA ratio without affecting 5-HIAA/5-HT ratios or NE content. 8-OH-DPAT alone had no effect on these parameters, although it decreased NE content in the prefrontal cortex in several experiments, and in the brainstem in one experiment. Significant decreases in NE content were observed in some brain regions following some of the stressors, but these changes were not generally affected by 8-OH-DPAT. Increases in the 5-HIAA/5-HT and DOPAC/DA ratios were also observed in some brain sites following some stressors, but these changes were not affected by 8-OH-DPAT except in the case of the increased 5-HIAA/5-HT ratio in the prefrontal cortex following the conditioned fear response. These results indicate that although 8-OH-DPAT is able to decrease plasma corticosterone responses following acoustic stress, conditioned fear, rHu-IL-1α, and cocaine administration, these effects do not appear to be related to an action of the 5-HT_1A agonist on biogenic amine metabolism. This observation indicates that the predominant effect of 8-OH-DPAT on adrenocortical responses is mediated at postsynaptic sites not involved in the regulation of cerebral biogenic amine metabolism.     

A biomimetic platform for studying root-environment interaction

Aims

Microstructure plays an important role in biological systems. Microstructural features are critical in the interaction between two biological organisms, for example, a microorganism and the surface of a plant. However, isolating the structural effect of the interaction from all other parameters is challenging when working directly with the natural system. Replicating microstructure of leaves was recently shown to be a powerful research tool for studying leaf-environment interaction. However, no such tool exists for roots. Roots present a special challenge because of their delicacy (specifically of root hairs) and their 3D structure. We aim at developing such a tool for roots.

Methods

Biomimetics use synthetic systems to mimic the structure of biological systems, enabling the isolation of structural function. Here we present a method which adapts tools from leaf microstructure replication to roots. We introduce new polymers for this replication.

Results

We find that Polyurethane methacrylate (PUMA) with fast UV curing gives a reliable replication of the tomato root surface microstructure. We show that our system is compatible with the pathogenic soilborne bacterium Ralstonia solanacearum.

Conclusions

This newly developed tool may be used to study the effect of microstructure, isolated from all other effects, on the interaction of roots with their environment.

     

Short interspersed DNA element-mediated detection of UVB-induced DNA damage and repair in the mouse genome, in vitro, and in vivo in skin.

We report a sensitive, SINE (Short Interspersed DNA Element)-mediated, PCR-based, DNA damage detection assay. Here, the SINE assay is used for detection of UVB-induced DNA damage and repair in cultured mouse cells and in vivo, in mouse skin. The unique feature of the SINE assay is its ability to support simultaneous amplification of multiple, random segments of genomic DNA. This can be accomplished due to the remarkable abundance, dispersion and conservation of SINEs in mammalian genomes. The most abundant SINEs in the mouse genome are the B1 elements, at a copy number of 50,000-80,000. Due to their strong sequence conservation, primers complementary to the B1 consensus sequence anneal to the majority of their targets in the genome. Consequently, long segments of genomic DNA located between pairs of B1 elements are efficiently amplified by PCR. Thus, in conjunction with the fact that many types of DNA adducts form blocks for thermostable polymerase, the B1 element anchored PCR makes a sensitive and versatile tool for assessing the overall integrity of the transcribed regions in mouse genome. We measured UVB-dose (0.1-3 kJ m-2) dependent formation of photoproducts in DNA from cultured cells, and after 20 h observed a substantial removal of damage at doses lower or equal to 0.6 kJ m-2. The sensitivity of detection of UVB-photoproducts formation and repair was compared to that of the conventional, single locus-targeting QPCR. Using the SINE assay we also have shown the distribution of UVB and UVC induced DNA adducts at a single nucleotide resolution within the B1 elements in mouse DNA. Lastly, we demonstrated that the sensitivity of the SINE assay is adequate for measurement of UVB-dose (1-6 kJ m-2) dependent formation and subsequent removal of photoproducts in vivo, in mouse skin.