Effects of vitamin A and insulin on the antioxidative state of diabetic rat heart: a comparison study with combination treatment

Because elevated oxidative stress may exacerbate cardiovascular complications of diabetes mellitus, the current study aimed to investigate the effects of treatment with either vitamin A, an antioxidant, or with insulin on lipid peroxidation products and antioxidant enzyme activities of diabetic rat heart. Also to evaluate whether a combination of vitamin A and insulin exerts more beneficial effects than treatment with each agent alone. Rats were made diabetic with a single injection of streptozotocin (STZ, 55 mg kg(-1) i.p.). Two days after STZ-injection, one group of diabetic rats was treated with vitamin A (retinol acetate, 30 mg kg(-1) day(-1) i.o.) for 12 weeks. A second group of diabetic rats was untreated for 6 weeks and then treated for another 6 weeks with insulin (8-10 IU rat(-1) day(-1) s.c.). Both therapies were applied to another group of diabetic rats for assessment of combined therapy with vitamin A plus insulin. Hearts from 12-week untreated diabetic animals showed about a four-fold increase in the level of thiobarbituric acid reactive substances (TBARS), indicative of increased lipid peroxidation. This was accompanied by approximately 100% increase in both catalase and glutathione peroxidase (GSHPx) enzyme activities. Therapy with insulin alone caused a small but significant improvement in plasma TBARS as well as GSHPx activities, but no significant change in plasma catalase in diabetic animals. Diabetes-induced disturbance in TBARS was almost completely prevented by vitamin A therapy. Although, a similar degree of activities for GSHPx was determined in diabetic animals treated with each agent alone, combination therapy was found to be more effective than single therapies in the recovery of GSHPx of diabetic heart. In contrast to insulin single therapy, vitamin A alone significantly prevented an increase in catalase activity of diabetic heart, and a combination of these agents did not supply any further benefit. Superoxide dismutase (SOD) activity was not found significantly different among the experimental groups. STZ-diabetes also resulted in less plasma retinol and retinol-binding protein (RBP), which was significantly improved by insulin single therapy while vitamin A used alone, failed to increase plasma retinol and RBP levels of diabetic animals. Our findings suggest that single therapy with insulin is unable to preclude oxidative reactions in diabetic heart to the same extent as obtained by vitamin A therapy alone, in spite of allowing recovery of normal growth rate and improved vitamin A metabolism in diabetic rats. A combination of insulin with vitamin A may provide more benefits than use of either agent alone in the treatment of general characteristics of diabetes and the maintenance of antioxidant defence of diabetic heart and thus in the reduction of peroxidative stress-induced cardiac injury.     

Implications for oxidative stress and astrocytes following 26S proteasomal depletion in mouse forebrain neurones

Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system, but the underlying mechanisms involved in neuroprotection and neurodegeneration remain unclear. Dysfunction of the ubiquitin proteasome system is one of the proposed hypotheses for the cause and progression of neuronal loss. We have performed quantitative two-dimensional fluorescence difference in-gel electrophoresis combined with peptide mass fingerprinting to reveal proteome changes associated with neurodegeneration following 26S proteasomal depletion in mouse forebrain neurones. Differentially expressed proteins were validated by Western blotting, biochemical assays and immunohistochemistry. Of significance was increased expression of the antioxidant enzyme peroxiredoxin 6 (PRDX6) in astrocytes, associated with oxidative stress. Interestingly, PRDX6 is a bifunctional enzyme with antioxidant peroxidase and phospholipase A₂ (PLA₂) activities. The PLA₂ activity of PRDX6 was also increased following 26S proteasomal depletion and may be involved in neuroprotective or neurodegenerative mechanisms. This is the first in vivo report of oxidative stress caused directly by neuronal proteasome dysfunction in the mammalian brain. The results contribute to understanding neuronal–glial interactions in disease pathogenesis, provide an in vivo link between prominent disease hypotheses and importantly, are of relevance to a heterogeneous spectrum of neurodegenerative diseases.     

Formulation of organic and inorganic hydrogel matrices for immobilization of β‐glucosidase in microfluidic platform

The aim of this study was to formulate silica and alginate hydrogels for immobilization of β‐glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate–polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized β‐glucosidase was loaded into glass–silicon–glass microreactors and catalysis of 4‐nitrophenyl β‐d ‐glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.     

Neuronal NADPH oxidase 2 regulates growth cone guidance downstream of slit2/robo2

NADPH oxidases (Nox) are membrane‐bound multi‐subunit protein complexes producing reactive oxygen species (ROS) that regulate many cellular processes. Emerging evidence suggests that Nox‐derived ROS also control neuronal development and axonal outgrowth. However, whether Nox act downstream of receptors for axonal growth and guidance cues is presently unknown. To answer this question, we cultured retinal ganglion cells (RGCs) derived from zebrafish embryos and exposed these neurons to netrin‐1, slit2, and brain‐derived neurotrophic factor (BDNF). To test the role of Nox in cue‐mediated growth and guidance, we either pharmacologically inhibited Nox or investigated neurons from mutant fish that are deficient in Nox2. We found that slit2‐mediated growth cone collapse, and axonal retraction were eliminated by Nox inhibition. Though we did not see an effect of either BDNF or netrin‐1 on growth rates, growth in the presence of netrin‐1 was reduced by Nox inhibition. Furthermore, attractive and repulsive growth cone turning in response to gradients of BDNF, netrin‐1, and slit2, respectively, were eliminated when Nox was inhibited in vitro. ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms involving Nox2 activation. We also investigated the possible relationship between Nox2 and slit2/Robo2 signaling in vivo. astray/nox2 double heterozygote larvae exhibited decreased area of tectal innervation as compared to individual heterozygotes, suggesting both Nox2 and Robo2 are required for establishment of retinotectal connections. Our results provide evidence that Nox2 acts downstream of slit2/Robo2 by mediating growth and guidance of developing zebrafish RGC neurons.     

A sensitive enzymeimmunoassay with a fluorimetric end-point for the determination of testosterone in female plasma and saliva

A fluorimetric enzymeimmunoassay has been developed having the sensitivity (50O fg/assay tube) required for determining testosterone concentrations in female plasma and saliva samples. The assay featured a solid-phase antiserum raised against an llα-hydroxytestosterone-11-hemisuccinate bovine serum albumin conjugate, an llα-hydroxytestosterone-11-hemisuccinate horseradish peroxidase conjugate as the “enzyme label” and p-hydroxyphenylacetic acid as the substrate for the development of fluorescence. Specificity was ensured by “extracting” testosterone from samples with a solid-phase anti testosterone-3-¦O-carboxymethyl¦-oxime serum. The assay was shown to satisfy accepted validation criteria providing results in good agreement with routine radioimmunoassay procedures in both plasma (r > 0.98, n = 28) and saliva (r > 0.99, n = 28). In saliva samples collected at 2 hourly intervals by normal healthy women (n = 5) testosterone concentrations showed a well defined circadian rhythm: the mean testosterone concentration in early morning samples (174 pmol/litre) fell by 83% in late evening collections. In healthy female volunteers (n = 7), mean salivary testosterone concentrations in samples collected daily throughout one complete cycle ranged from 5O to 218 pmol/litre. Following dexamethasone administration testosterone concentrations in plasma fell by approximately 50% and salivary concentrations were undetectable after one hour. This enzymeimmunoassay may be useful in studies of female infertility.     

Genetic and epigenetic effects of salinity on in vitro growth of barley

Morphological, physiological and molecular changes were investigated in in vitro salt-stressed barley (Hordeum vulgare L. cv. Tokak). Mature embryos were cultured in Murashige and Skoog medium containing 0 (control), 50 and 100 mM NaCl for 20 days. Both concentrations inhibited shoot growth, decreased fresh weight and protein content, and increased SOD (EC 1.15.1.1) activity in a dose-dependent manner. The lower concentration increased root growth. Salinity caused nucleotide variations in roots, but did not affect shoot DNAs. The higher concentration caused methylation changes, mainly hypermethylation in shoots. This is the first study on genetic and epigenetic effects of salinity in barley.     

Two complementary strand-specific termination sites for adenovirus DNA replication

Adenovirus type 2 DNA, specifically labeled at the termini for DNA replication, was prepared by isolation of viral DNA molecules which were completed during short pulses with ³H-thymidine. The distribution of radioactivity in the two complementary strands at the termini for DNA replication was determined by liquid phase hybridization and gel electrophoresis. At the right-hand terminus, nearly all radioactivity was found in the viral h strand, whereas at the left-hand terminus, most radioactivity was confined to the viral I strand. The results suggest that both molecular ends serve as origins and termini for replication of adenovirus type 2 DNA.