Mechanisms involved in the development of diabetic retinopathy induced by oxidative stress

Background: Diabetic retinopathy (DR) is one of the main complications in patients with diabetes and has been the leading cause of visual loss since 1990. Oxidative stress is a biological process resulting from excessive production of reactive oxygen species (ROS). This process contributes to the development of many diseases and disease complications. ROS interact with various cellular components to induce cell injury. Fortunately, there is an antioxidan t system that protects organisms against ROS. Indeed, when ROS exceed antioxidant capacity, the resulting cell injury can cause diverse physiological and pathological changes that could lead to a disease like DR.

Objective: This paper reviews the possible mechanisms of common and novel biomarkers involved in the development of DR and explores how these biomarkers could be used to monitor the damage induced by oxidative stress in DR, which is a significant complication in people with diabetes.

Conclusion: The poor control of glucemy in pacients with DB has been shown contribute to the development of complications in eyes as DR.     

Spermine stimulation of phospholipase C fromCatharanthus roseus transformed roots

Polyamines (Pas) are aliphatic amines that are ubiquitous in all living organisms and regulate a broad spectrum of physiological processes. It has been suggested that they can act through a signal transduction pathway. Using Catharanthus roseus hairy roots as a model we determined the levels of Pas throughout a culture cycle. We found that there is a peak in the intracellular concentration of Pas during the first six days of culture. The effect of Pas on phospholipase C (PLC) activity was also investigated. Putrescine, spermidine and spermine were added in vitro to the PLC assay. Putrescine did not modify PLC activity; spermidine inhibited the enzyme but at very high, non-physiological concentrations; and spermine increased the PLC activity four-fold at physiological concentrations. Our results suggest that spermine could regulate root growth by regulating the PLC signal transduction mechanism.     

Fast drops: a high-throughput approach for setting up protein crystal screens.

There is significant demand to rapidly obtain protein structure information for both structural genomics and drug discovery applications. To meet this demand, all steps in the process of determining protein structure by X-ray crystallography need to be optimized and streamlined with high-throughput methodologies. This communication describes a method that brings high-throughput technology to protein crystallization in both manual and automated modes, suitable for virtually every crystallography laboratory.     

Testosterone increases neurotoxicity of glutamate in vitro and ischemia-reperfusion injury in an animal model.

Increasing evidence has demonstrated striking sex differences in the outcome of neurological injury. Whereas estrogens contribute to these differences by attenuating neurotoxicity and ischemia-reperfusion injury, the effects of testosterone are unclear. The present study was undertaken to determine the effects of testosterone on neuronal injury in both a cell-culture model and a rodent ischemia-reperfusion model. Glutamate-induced HT-22 cell-death model was used to evaluate the effects of testosterone on cell survival. Testosterone was shown to significantly increase the toxicity of glutamate at a 10 microM concentration, whereas 17beta-estradiol significantly attenuated the toxicity at the same concentration. In a rodent stroke model, ischemia-reperfusion injury was induced by temporal middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h. To avoid the stress-related testosterone reduction, male rats were castrated and testosterone was replaced by testosterone pellet implantation. Testosterone pellets were removed at 1, 2, 4, or 6 h before MCAO to determine the duration of acute testosterone depletion effects on infarct volume. Ischemic lesion volume was significantly decreased from 239.6 +/- 25.9 mm(3) in control to 122.5 +/- 28.6 mm(3) when testosterone pellets were removed at 6 h before MCAO. Reduction of lesion volume was associated with amelioration of the hyperemia during reperfusion. Our in vitro and in vivo studies suggest that sex differences in response to brain injury are partly due to the consequence of damaging effects of testosterone.     

The effects of nerve growth factor upon the neuropeptide content of the suprachiasmatic nucleus of rats withdrawn from ethanol are mediated by the nucleus basalis magnocellularis

It has been previously shown that withdrawal from alcohol decreases the synthesis and expression of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN), and that the infusion of NGF over 1 month completely restores these changes. Because SCN neurons do not express TrkA, NGF might have exerted its effects either through direct signalling of the neurons via p75^NTR or by enhancing the activity of the cholinergic afferents to the SCN, which arise from the nucleus basalis magnocellularis (NBM). The observation that the infusion of NT-3 to withdrawn rats does not elicit any change in neuropeptide expression in the SCN suggests that ACh might be implicated in this process, a hypothesis that we have attempted to clarify in this study. For this purpose we destroyed, with quinolinic acid, the NBM of rats withdrawn from ethanol and later infused them with NGF over a period of 13 days. The total number and the somatic volume of SCN neurons immunoreactive for VP and VIP were stereologically estimated. No differences were found in the total number of neurons between quinolinic-injected NGF-treated withdrawn animals and intact withdrawn rats. However, the somatic volume of SCN neurons from quinolinic-injected animals was significantly reduced relative to control and withdrawn rats. The present results unequivocally demonstrate that the trophic effects exerted by NGF upon SCN neurons do not depend on direct neuronal signalling. Instead, they are indirect and, according to our results, NBM neurons, whose axons give rise to a cholinergic projection to the SCN, seem to be essential for eliciting those effects.     

Leptin and Altitude in the Cardiovascular Diseases

Objective: The lower mortality from coronary ischemic disease in populations living at high altitude has been related to an increase of high‐density lipoprotein (HDL)‐cholesterol at altitude. Leptin has been proposed as a cardiovascular risk factor. We investigated whether leptin varies according to the altitude at which people live. Research Methods and Procedures: This was a cross‐sectional study of the first 889 people enrolled in a cohort study in the Canary Islands, Spain. The relationship among serum leptin, altitude, obesity, and other cardiovascular risk factors was analyzed by bivariate and multivariate tests. Results: Leptin levels showed an inverse correlation to altitude expressed in meters (r = ?0.10). Obese subjects had this leptin‐altitude association (r = ?0.19), but they also had a direct correlation of leptin to HDL‐cholesterol (r = 0.27) and an inverse correlation of leptin to the total cholesterol‐to‐HDL‐cholesterol ratio (r = ?0.34), triglycerides (r = ?0.29), apolipoprotein B (r = ?0.21), and glycemia (r = ?0.19). Nonobese subjects had only the leptin‐altitude association (r = ?0.11). The final regression model included altitude as predictor. Other associated variables were gender, physical activity, BMI, age, smoking (reducing leptin independently of BMI), alcohol, heart rate, and income. Discussion: Serum leptin level decreases when altitude increases, and this association could help to explain the lower cardiovascular mortality rate at high altitude. However, because in obese subjects there is a direct association of leptin with HDL‐cholesterol and an inverse association with the lipid atherogenic fractions, we suggest the hypothesis of different roles for bound and free leptin, with free leptin being a cardiovascular protective factor in obese people.     

Patterns of diversity of the north-eastern Atlantic blenniid fish fauna (Pisces: Blenniidae)

This paper presents an analysis of the distributional patterns of blenniids (Pisces: Blenniidae) in the north‐eastern Atlantic. Two peaks of species diversity were found, both in terms of number of species and number of endemics: one in the tropical African coast and another in the Mediterranean Sea. A cluster analysis of similarity values (Jaccard coefficient) among the eastern Atlantic zoogeographical areas, revealed the following groups: a north temperate group, a tropical group formed by the tropical African coast and Mauritania, another group formed by the islands of Cape Verde, a south temperate group (South Africa), and a southern Atlantic group formed by the islands of Ascension and St Helena. Within the north temperate group, the subgroups with higher similarities were: Azores and Madeira, Canary Islands and Morocco, and the Mediterranean and the Atlantic coast of the Iberian Peninsula. Based on affinity indices, the probable directions of faunal flows were inferred. The tropical coast of Africa and the Mediterranean emerged from this analysis as probable speciation centres of the north‐eastern Atlantic blenniid fauna. The Mediterranean may have also acted as a refuge during glacial periods.     

The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor.

Ligand-induced receptor oligomerization is an established mechanism for receptor-tyrosine kinase activation. However, numerous receptor-tyrosine kinases are expressed in multicomponent complexes with other receptors that may signal independently or alter the binding characteristics of the receptor-tyrosine kinase. Nerve growth factor (NGF) interacts with two structurally unrelated receptors, the Trk A receptor-tyrosine kinase and p75, a tumor necrosis factor receptor family member. Each receptor binds independently to NGF with predominantly low affinity (K(d) = 10(-9) m), but they produce high affinity binding sites (K(d) = 10(-11) m) upon receptor co-expression. Here we provide evidence that the number of high affinity sites is regulated by the ratio of the two receptors and by specific domains of Trk A and p75. Co-expression of Trk A containing mutant transmembrane or cytoplasmic domains with p75 yielded reduced numbers of high affinity binding sites. Similarly, co-expression of mutant p75 containing altered transmembrane and cytoplasmic domains with Trk A also resulted in predominantly low affinity binding sites. Surprisingly, extracellular domain mutations of p75 that abolished NGF binding still generated high affinity binding with Trk A. These results indicate that the transmembrane and cytoplasmic domains of Trk A and p75 are responsible for high affinity site formation and suggest that p75 alters the conformation of Trk A to generate high affinity NGF binding.     

Characterization of nitroproteome in neuron-like PC12 cells differentiated with nerve growth factor: identification of two nitration sites in alpha-tubulin

Nitric oxide (NO) is a precursor of reactive nitrating species, peroxynitrite and nitrogen dioxide, which modify proteins to generate oxidized species such as 3-nitrotyrosine that has been used as a hallmark of peroxynitrite-mediated oxidative stress on proteins. In the last few years however, a growing body of evidence indicates that NO also regulates a myriad of physiologic responses by modifying tyrosine residues. Looking for the molecular event triggered by NO in nerve growth factor (NGF)-induced neuronal differentiation, we recently reported that in differentiating PC12 cells, the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins, and alpha-tubulin is the major target. In the present work, we focus on the investigation of the sites of tyrosine nitration in alpha-tubulin purified by two-dimensional gel electrophoresis following anti-alpha-tubulin immunoprecipitation of protein extract from NGF-treated PC12 cells. Using Western blotting and matrix-assisted laser desorption/ionization-time of flight analysis, we show for the first time, both in vivo and in vitro, that nitration can occur on alpha-tubulin at sites other than the C-terminus and we positively identify Tyr 161 and Tyr 357 as two specific amino acids endogenously nitrated.