Almudena Crooke

Failure to increase glucose consumption through the pentose-phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene- deleted mouse embryonic stem cells subjected to oxidative stress

Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells ( G6pd delta), obtained by transient Cre recombinase expression in a G6pd -loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 1.1.1.42). These G6pd delta cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pd delta cells show a high ratio of NADPH to NADP(+) and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pd delta but not for wild-type ES cells, NADPH and GSH in G6pd delta cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP(+)] ratio, does not occur in G6pd delta cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.

Nucleotides in ocular secretions: Their role in ocular physiology

The eye is the sense organ that permits the detection of light owing to the existence of a sophisticated neuronal array, called the retina, which is responsive to photons. The correct functioning of this complex system requires the coordination of several intraocular structures that ultimately permit the perfect focusing of images on the neural retina. Light has to pass through different media: the tear, the cornea, aqueous humour, lens, and vitreous humour before it reaches the retina. Moreover, the composition and structure of some of these media can change due to several physiological mechanisms. Nucleotides are active components of the humours bathing relevant ocular structures. The tear contains nucleotides and dinucleotides that control the process of tearing, wound healing and protects of superficial infections. In the inner eye, the aqueous humour also presents a collection of mono and dinucleotides that affect pupil contraction, aqueous humour production and accommodation. Behind the lens and between this structure and the retina the vitreous humour can modify the physiology of the retinal cells, mostly the ganglion cells. By investigating the actions of nucleotides and dinucleotide present in the ocular humours we will be able not only to understand the functioning of the ocular structures but also to develop new pharmacological therapies for pathologies such as dry eye, glaucoma or retinal detachment.

Corneal re-epithelialization stimulated by diadenosine polyphosphates recruits RhoA/ROCK and ERK1/2 pathways.

PURPOSE To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap(4)A and Ap(3)A. METHODS In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap(4)A or Ap(3)A 100 microM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 microM, U0126 100 microM, Y27632 100 nM, and (-)-blebbistatin 10 microM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap(4)A and Ap(3)A (100 microM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap(4)A or Ap(3)A (100 microM) with U0126 and Y27632 (100 nM). RESULTS In the presence of Ap(4)A, U0126, Y27632, AG1478, and (-)-blebbistatin, reduced the migration rate compared to the effect of Ap(4)A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap(4)A, respectively). In the presence of Ap(3)A 100 microM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap(3)A alone, whereas AG1478 and (-)-blebbistatin (P < 0.0001 versus Ap(3)A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap(4)A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. CONCLUSIONS The activation of the Ap(4)A/P2Y(2) receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap(3)A/P2Y(6) receptor signals only the MAPK pathway.

Sympathetic nervous system modulates the ocular hypotensive action of MT2‐melatonin receptors in normotensive rabbits

Abstract:  The aim of this study was to investigate the hypotensive effect of the melatonin analogue, N‐butanoyl‐2‐(2‐methoxy‐6H‐isoindolo[2,1‐a]indol‐11‐yl)ethanamine (IIK7), through MT2‐melatonin receptors and the involvement of the sympathetic nervous system in this action in New Zealand rabbit eyes. The topical application of melatonin or IIK7 produced a reduction in intraocular pressure of 20.2 ± 5.3% and 38.5 ± 3.2% respectively. This effect was concentration‐dependent; it was blocked by selective MT2 receptor antagonists and was severely diminished after chemical sympathectomy. Immunohistochemistry and western blot analysis showed the ciliary processes as the site of this action and no co‐localization of MT2‐melatonin receptor with the sympathetic nervous system was observed. The β‐adrenergic agonists, terbutaline and salbutamol, potentiated the hypotensive effect of IIK7 reducing intraocular pressure (IOP) 41.75 ± 4.26% and 44.7 ± 5.6% respectively. Also, IIK7 in presence of the nonspecific protein phosphatase inhibitor okadaic acid, lowered IOP 32.2 ± 4.5% and in presence of forskolin plus 3‐isobutyl‐1‐methylxanthine decreased IOP in 32.2 ± 5.47%. These data suggest that the melatonin agonist IIK7 reduces intraocular pressure by acting through MT2‐melatonin receptors presumably decreasing aqueous humour formation. Also, in the presence of β‐adrenoceptor agonists MT2‐melatonin receptors activity increase their ability to reduce IOP.

Transient silencing of Plasmodium falciparum bifunctional glucose‐6‐phosphate dehydrogenase− 6‐phosphogluconolactonase

The bifunctional enzyme glucose‐6‐phosphate dehydrogenase‐6‐phosphogluconolactonase (G6PD‐6PGL) found in Plasmodium falciparum has unique structural and functional characteristics restricted to this genus. This study was designed to examine the effects of RNA‐mediated PfG6PD‐6PGL gene silencing in cultures of P. falciparum on the expression of parasite antioxidant defense genes at the transcription level. The highest degree of G6PD‐6PGL silencing achieved was 86% at the mRNA level, with a recovery to almost normal levels within 24 h, indicating only transient diminished expression of the PfG6PD‐6PGL gene. PfG6PD‐6PGL silencing caused arrest of the trophozoite stage and enhanced gametocyte formation. In addition, an immediate transcriptional response was shown by thioredoxin reductase suggesting that P. falciparum G6PD‐6PGL plays a physiological role in the specific response of the parasite to intracellullar oxidative stress. P. falciparum transfection with an empty DNA vector also promoted intracellular stress, as determined by mRNA up‐regulation of antioxidant genes. Collectively, our findings point to an important role for this enzyme in the parasites infection cycle. The different characteristics of G6PD‐6PGL with respect to its homologue in the host make it an ideal target for therapeutic strategies.

5‐MCA‐NAT does not act through NQO2 to reduce intraocular pressure in New‐Zealand white rabbit

Abstract:  Solid data support the idea that the MT3 melatonin binding site is an enzyme, quinone reductase 2 (NQO2), rather than a membrane melatonin receptor. However, the melatonin analogue, 5‐methoxycarbonylamino‐N‐acetyltryptamine (5‐MCA‐NAT), reduces intraocular pressure (IOP) via MT3 melatonin receptors. Therefore, the aim of this work was to test whether the melatonin binding site, MT3, is indeed the enzyme NQO2 in New Zealand rabbit eyes. To investigate this, the action of several substrates and inhibitors for NQO2 was compared to 5‐MCA‐NAT in their ability to modify IOP. Also, the effect of 5‐MCA‐NAT on IOP produced after NQO2 silencing by means of a siRNA was determinated. Altogether, the results led us to conclude that the in vivo effect of the MT3 ligand 5‐MCA‐NAT on IOP is not mediated by the enzyme NQO2, suggesting the existence of another melatonin receptor.

Signs and Symptoms of Dry Eye in Keratoconus Patients: A Pilot Study

Abstract Purpose: To compare signs and symptoms of dry eye in keratoconus (KC) patients versus healthy subjects. Methods: A total of 15 KC patients (KC group, n = 15 eyes) and 16 healthy subjects (control group, 16 eyes) were enrolled in this study. The Schirmer I test with no anesthetic, tear break-up time (TBUT), corneal staining characteristics, and ocular surface disease index (OSDI) scores were evaluated for both groups. Impression cytology, combined with/scanning laser confocal microscopy (LCM), was performed to evaluate goblet cell density, mucin cloud height (MCH), and goblet cell layer thickness (CLT). Finally, tear concentrations of di-adenosine tetraphosphate (Ap4A) were assessed. Results were statistically analyzed using Shapiro–Wilk and non-parametric Wilcoxon rank sum tests. Statistical significance was set at p < 0.05. Results: KC patients had lower tear volumes and greater corneal staining than did healthy subjects (p < 0.05). OSDI scores were 44.96 ± 8.65 and 17.78 ± 6.50 for the KC and control groups, respectively (p < 0.05). We found no statistically significant differences in TBUT between groups. Impression cytology revealed lower goblet cell densities in KC group patients versus control group subjects (84.88 ± 32.98 and 128.88 ± 50.60 cells/mm,2 respectively, p < 0.05). There was a statistically significant reduction in MCH and CLT in KC group patients compared with control group subjects. Ap4A tear concentrations were higher in KC group patients than in control group subjects (2.56 ± 1.10 and 0.15 ± 0.12 µM, respectively, p < 0.05). Conclusions: The parameters evaluated in this study indicate that KC patients suffer greater symptoms of dry eye and greater tear instability, primarily due to the decreased mucin production in their tears, than do healthy patients with no KC

Silencing of P2Y2 receptors reduces intraocular pressure in New Zealand rabbits

P2 receptors are involved in the regulation of ocular physiological processes like intraocular pressure (IOP). In the present study, the involvement of P2Y2 receptors in the hypertensive effect of nucleotides was investigated by use of antagonists and of a siRNA designed for the P2Y2 receptor.

Long-Term Follow-Up of Donor Chimerism and Tolerance After Human Liver Transplantation

We aimed to quantify peripheral donor chimerism (DC) and to analyze its association with graft and recipient outcome. Forty‐two liver transplant recipients and their respective donors were studied, providing a total of 148 posttransplantation serum samples. DC was assessed with real‐time quantitative polymerase chain reaction (qPCR) to detect polymorphic markers. DC did not decrease with time post‐transplantation and was higher in child recipients versus adults and in recipients of deceased donor liver transplants versus recipients of live donor liver transplants. Higher levels of DC were detected in Rh‐positive blood group donors, in O blood group recipients versus A blood group recipients, and in recipients with hepatitis C virus versus recipients with alcoholic cirrhosis. High DC was associated with patients with organ damage due to recurrent disease and rejection. Stable, high levels of DC, in the absence of other major clinical events, may thus be a marker of transplantation tolerance, and this knowledge may help to tailor immunosuppressive treatment. In conclusion, qPCR is a useful technique for DC follow‐up in liver transplantation, although the evolution of DC levels should be analyzed in accordance with the clinical outcome of the patient. Liver Transpl 15:581–591, 2009.

Requirement of intact sympathetic transmission for the ocular hypotensive effects of melatonin and 5-MCA-NAT

Melatonin and its analogue, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), potently reduce intraocular pressure, and may be good candidates for the treatment of ocular hypertension and glaucoma. After chemical sympathectomy by reserpine or 6-hydroxydopamine, the hypotensive effects of melatonin and 5-MCA-NAT are severely inhibited. This indicates that the sympathetic nervous system is involved in the production and drainage of aqueous humour by the ciliary body and trabecular meshwork, and that it mediates the effects of melatonin and its analogue, 5-MCA-NAT.