Delivery of glutamine synthetase gene by baculovirus vectors: a proof of concept for the treatment of acute hyperammonemia.

Hyperammonemia, a condition present in patients with urea cycle disorders (UCDs) or liver diseases, can cause neuropsychiatric complications, which in the worst cases result in brain damage, coma or death. Diverse treatments exist for the treatment of hyperammonemia, but they have limited efficacy, adverse effects and elevated cost. Gene therapy is a promising alternative that is explored here. A baculovirus, termed Bac-GS, containing the glutamine synthetase (GS) gene was constructed for the in vitro and in vivo treatment of hyperammonemia. Transduction of MA104 epithelial or L6 myoblast/myotubes cells with Bac-GS resulted in a high expression of the GS gene, an increase in GS concentration, and a reduction of almost half of exogenously added ammonia. When Bac-GS was tested in an acute hyperammonemia rat model by intramuscularly injecting the rear legs, the concentration of ammonia in blood decreased 351 µM, in comparison with controls. A high GS concentration was detected in gastrocnemius muscles from the rats transduced with Bac-GS. These results show that gene delivery for overexpressing GS in muscle tissue is a promising alternative for the treatment of hyperammonemia in patients with acute or chronic liver diseases and hepatic encephalopathy or UCD.

Melatonin-induced methylation of the ABCG2/BCRP promoter as a novel mechanism to overcome multidrug resistance in brain tumour stem cells.

BACKGROUND: Current evidence indicates that a stem cell-like sub-population within malignant glioblastomas, that overexpress members of the adenosine triphosphate-binding cassette (ABC) family transporters, is responsible for multidrug resistance and tumour relapse. Eradication of the brain tumour stem cell (BTSC) compartment is therefore essential to achieve a stable and long-lasting remission. METHODS: Melatonin actions were analysed by viability cell assays, flow cytometry, quantitative PCR for mRNA expression, western blot for protein expression and quantitative and qualitative promoter methylation methods. RESULTS: Combinations of melatonin and chemotherapeutic drugs (including temozolomide, current treatment for malignant gliomas) have a synergistic toxic effect on BTSCs and A172 malignant glioma cells. This effect is correlated with a downregulation of the expression and function of the ABC transporter ABCG2/BCRP. Melatonin increased the methylation levels of the ABCG2/BCRP promoter and the effects on ABCG2/BCRP expression and function were prevented by preincubation with a DNA methyltransferase inhibitor. CONCLUSION: Our results point out a possible relationship between the downregulation of ABCG2/BCRP function and the synergistic toxic effect of melatonin and chemotherapeutic drugs. Melatonin could be a promising candidate to overcome multidrug resistance in the treatment of glioblastomas, and thus improve the efficiency of current therapies.

[A proposed new target for deep brain stimulation in obsessive-compulsive disorder]. / Propuesta de una nueva diana para la estimulación cerebral profunda en el trastorno obsesivo-compulsivo.

INTRODUCTION: The obsessive-compulsive disorder (OCD) has an incidence in general population of 1.5-3%. If we consider as a positive respond a diminution of the 25-35% in the symptoms of OCD according to the Y-BOCS, and we add the cognitive-behavioral therapy to the pharmacological treatment, only a 40-60% of treated patients would have significant improvement and a 10% of patients with OCD, would be refractory to all type of medical treatment. DEVELOPMENT: Current neurosurgical techniques for resistant cases of OCD interrupt the connections between the frontal lobes and subcortical structures (cingulotomy, capsulotomy). These techniques are ablative and irreversible. It shows the importance of finding a less aggressive technique with better clinical results. Deep brain stimulation (DBS) is an alternative to traditional neurosurgery based in neuromodulation methods. It's considered that the physiopathology of the OCD consists of a dysfunction of the direct and indirect vias that control the extrapiramidal limbic circuit. On the other hand, it had been obtained positive results after DBS of the subthalamic nucleus of three patients with Parkinson's disease and OCD. CONCLUSION: This article has as target the demonstration that bilateral DBS of the limbic part of the subthalamic nucleus is an alternative for the treatment of refractory OCD.

Localization of the lipid receptors CD36 and CLA-1/SR-BI in the human gastrointestinal tract: towards the identification of receptors mediating the intestinal absorption of dietary lipids.

The scavenger receptors CLA-1/SR-BI and CD36 interact with native and modified lipoproteins and with some anionic phospholipids. In addition, CD36 binds/transports long-chain free fatty acids. Recent biochemical evidences indicates that the rabbit CLA-1/SR-BI receptor can be detected in enterocytes, and previous studies showed the presence of mRNA for both CLA-1/SR-BI and CD36 in some segments of the intestinal tract. These findings prompted us to study their respective localization and distribution from the human stomach to the colorectal segments, using immunohistochemical methods. Their expression in the colorectal carcinoma-derived cell line Caco-2 was analyzed by Northern blotting. In the human intestinal tract, CLA-1/SR-BI was found in the brush-border membrane of enterocytes from the duodenum to the rectum. However, CD36 was found only in the duodenal and jejunal epithelium, whereas enterocytes from other intestinal segments were not stained. In the duodenum and jejunum, CD36 co-localized with CLA-1/SR-BI in the apical membrane of enterocytes. The gastric epithelium was immunonegative for both glycoproteins. We also found that CLA-1/SR-BI mRNA was expressed in Caco-2 cells and that its expression levels increased concomitantly with their differentiation. In contrast, the CD36 transcript was not found in this colon cell line, in agreement with the absence of this protein in colon epithelium. The specific localization of CLA-1/SR-BI and CD36 along the human gastrointestinal tract and their ability to interact with a large variety of lipids strongly support a physiological role for them in absorption of dietary lipids.

Gene transfer into cystic fibrosis airway epithelial cells.

Gene transfer into airway epithelial cells becomes a particularly motivating goal as far as cystic fibrosis (CF) is concerned. As mentioned in Chapter 15 , approx 90% of deaths caused by this devastating disease are the result of infections of the respiratory tract owing to dysfunction of the Cl(-) transport in airway epithelial cells. Efficient transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium of CF patients in vivo is one of the current challenges of gene therapists, and much is being made in that direction in the United States, Europe, and Argentina. The airway epithelium has a very slow turnover, with presumably only l-2% of cells at division at a given time. Therefore, retroviruses are not the most suitable vector system for gene transfer of the CF gene (1). Adenovirus vectors, adeno-associated virus vectors, or liposomes are being tested in the gene therapy clinical trials currently going on (2, 3). In vitro CF gene transfer into airway epithelial cells has relevance in the optimization of gene transfer vectors, of CFTR activity tests, of endogenous as well as vector-produced CFTR-mRNA analysis, and so forth, related to the in vivo application. In the following sections, analysis will restrict itself to in vitro gene transfer approaches.

Genetic analysis of cystic fibrosis airway epithelial cells.

More than 500 different mutations have been described to date on the cystic fibrosis (CF) gene (1). The most frequent mutation is the so-called ΔF508 mutation, which accounts for 30-50%;of CF chromosomes in southern European countries and for 50-80%; of CF chromosomes in the United States, Canada, Argentina, and central and northern European countries.

Liver alcohol dehydrogenase immobilized on polyvinylidene difluoride.

A physical method for immobilization of liver alcohol dehydrogenase (ADH) by hydrophobic adsorption onto a supporting membrane of polyvinylidene difluoride (PVDF) was performed. Simultaneously, a physicochemical characterization of the immobilized enzyme regarding its kinetic behaviour was performed. The activity/pH profile observed points to an effect of pH on activity that is completely different from the case of ADH in solution. The disturbance in the typical bell-shaped profile owing to the fact that the enzyme was immobilized is explained on the basis of a potent limitation to the diffusion of the protons in the support. The findings of the present work also reveal the existence of an effect that limits free external diffusion of the substrate towards and/or the product from the support; this effect seems to be the determinant of the overall rate of the enzymatic reaction and is thus of great importance in the effective kinetic behaviour (v([S])) of immobilized ADH, whose kinetic behaviour is complex (non-Michaelian), as may be seen from the lack of linearity observed in the corresponding double reciprocal and Eadie-Hofstee plots. By non-linear regression numerical analysis of the v([S]) data and application of the F-test for model discrimination, the minimum rate equation necessary to describe the intrinsic kinetic behaviour of PVDF-immobilized ADH proved to be one of the polynomial quotient type of degree 2:2 (in substrate concentration).

Elevated arsenic and manganese in groundwaters of Murshidabad, West Bengal, India.

High levels of geogenic arsenic (As) and manganese (Mn) in drinking water has led to widespread health problems for the population of West Bengal, India. Here we delineate the extent of occurrences of As and Mn in Murshidabad, where the contaminated aquifers occur at shallow depths between 35 and 40 m and where access to safe drinking water is a critical issue for the local population. A total of 78 well-water samples were taken in 4 blocks on either side of the river Bhagirathi: Nabagram and Kandi (west, Pleistocene sediments), Hariharpara and Beldanga (east, Holocene sediments). High As, total iron (FeT) and low Mn concentrations were found in waters from the Holocene gray sediment aquifers east of the river Bhagirathi, while the opposite was found in the Pleistocene reddish-brown aquifer west of the river Bhagirathi in Murshidabad. Speciation of As in water samples from Holocene sediments revealed the dominant species to be As(III), with ratios of As(III):AsT ranging from 0.55 to 0.98 (average 0.74). There were indications from saturation index estimations that Mn solubility is limited by the precipitation of MnCO3. Tubewells from high As areas in proximity to anthropogenic waste influx sources showing high molar Cl/Br ratios, low SO4(2-) and low NO3(-) demonstrate relatively lower As concentrations, thereby reducing As pollution in those wells. Analyses of core samples (2 in each of the blocks) drilled to a depth of 45 m indicate that there is no significant variation in bulk As (5-20mg/kg) between the Holocene and Pleistocene sediments, indicating that favorable subsurface redox conditions conducive to mobilization are responsible for the release of As. The same applies to Mn, but concentrations vary more widely (20-2000 mg/kg). Sequential extraction of Holocene sediments showed As to be associated with 'specifically sorbed-phosphate-extractable' phases (10-15%) and with 'amorphous and well crystalline Fe-oxyhydroxide' phases (around 37%) at As-contaminated well depths, suggesting that the main As release mechanisms could be either competitive ion exchange with PO4(3-), or the dissolution of Fe oxyhydroxides. In the Pleistocene sediments Mn is predominantly found in the easily exchangeable fraction.

[Pathogenetic bases of epileptogenesis in cerebral cavernomas]. / Bases patogenicas de la epileptogenesis en los cavernomas cerebrales.

INTRODUCTION. Brain cavernoma are a type of arteriovenous malformation that clinically presenting seizures, neurological deficit or bleeding. Hypoxia, neoangiogenesis and metalloproteasas seems to be involved in seizures physiopathology. Our study aims to assess this potential relation by immunohistochemical methods, analyzing hypoxia inducible factor (HIF-1alpha) and metalloproteasa (MMP-9) in tissue surrounding cavernoma. PATIENTS AND METHODS. We selected 17 consecutive cases anatomopathologically diagnosed as cavernoma during 9 years. Immunohistochemical staining was performed for HIF-1alpha and MMP-9. We evaluated the relation between seizures and the scale of uptake of different tissues surrounding cavernoma. RESULTS. Cases with seizures had HIF-1alpha positive uptake in vascular endothelium in 31%, 17% in fibrous tissue and 34% in inflammatory tissue. Besides, it also shows MMP-9 positive uptake in vascular endothelium in 86%, 100% in fibrous tissue and 43% of brain tissue. Statistical analysis by chi-square and odds ratio shows a positive trend towards seizures and the presence of HIF-1alpha and MMP-9 in vascular tissue, fibrous tissue and brain tissue, but no for inflammatory tissue. CONCLUSION. HIF-1alpha and MMP-9, valued by immunohistochemical methods, are related to complications as seizures.

[Review of the target for the deep brain stimulation of chronic cluster headache]. / Revision de la diana para la estimulacion cerebral profunda de la cefalea en racimos cronica.

Cluster headache is included in the group of trigeminal autonomic cephalalgias. Although the pathophysiology of cluster headache has not yet been sufficiently established, the theory of a central origin tells us that this headache is produced by hypothalamic dysfunction. More than 50 patients have been treated with deep brain stimulation of the posterior nucleus of the hypothalamus from 2001. The results show clinical improvement in more than 60% of the cases, opening a promising issue for the treatment of the cluster headache persistent after medical treatment. The surgical target that have been used until now is based on the origin of the cluster headache in the hypothalamic dysfunction. Nevertheless, It has still some open questions as the lack of proving the posterior nucleus of the hypothalamus is the real origin of the cluster headache, the lack of consensus about the anatomy of the surgical target and the variability of the structures stimulated with the surgery. The aim of this article is a review of the target used and propose another surgical target based on physiopathological concepts to explain the improvement with the deep brain stimulation in these patients.

[Surgical treatment of the stroke in the middle cerebral artery]. / Tratamiento quirúrgico del ictus de la arteria cerebral media.

INTRODUCTION: Decompressive craniectomy increases the survival rate in cases of malignant middle cerebral artery (MCA) stroke. The imaging and clinical signs that predict a malignant progression of stroke of the MCA are analysed, together with factors associated with a poorer prognosis. PATIENTS AND METHODS: The study involved 30 patients, who were divided into three groups: patients who had undergone surgery, and patients who had not undergone surgery but were admitted to intensive care or to neurology wards. The surgical procedure consisted in creating a bone window with a diameter of at least 10 cm and a dural opening. The initial evaluation of the patient was performed using the Glasgow scale and the National Institute of Health stroke scale; follow-up was carried out using the modified Rankin scale, the Barthel index and the Glasgow Outcome Scale at six months. RESULTS: Younger patients have a better functional prognosis than those over 60 years of age. A deviation of more than 10 mm from the mean line is associated with a poorer prognosis, as are volumes of infarcted tissue above 350 cm3. Lower scores on the Glasgow scale on admission are associated with a poorer prognosis for survival and a higher number of sequelae, as well as their reduction during hospitalisation. CONCLUSIONS: Age conditions the presence of sequelae in these patients. The presence of clinical signs of herniation (anisocoria, lower initial score or important drop on the Glasgow scale) and imaging signs (displacement of the mean line, volume of infarcted tissue) imply a poorer prognosis. Early surgery in those patients in whom it is indicated reduces the number of sequelae and increases the rate of survival.

Complications in subthalamic nucleus stimulation surgery for treatment of Parkinson's disease. Review of 272 procedures.

BACKGROUND: Deep brain stimulation (DBS) is a surgical technique used to alleviate symptoms in patients with advanced Parkinson's disease (PD). It is a reversible procedure and its effect is based on electrical modulation of the nervous system and has considerable advantages in morbidity-mortality when compared to lesion techniques such as thalamotomy and/or pallidotomy. The objective was to evaluate the adverse events during the surgical placement of leads in the subthalamic nucleus for the treatment of Parkinson's disease. METHODS: A retrospective data collection was made in a total of 130 patients in whom we performed 272 procedures for the implant of leads in the subthalamic nucleus between May 1998 and December 2005. All the patients were operated by the same surgeon, in the same institution and with the same surgical methodology. The complications under evaluation were: aborted procedure, misplaced leads, intracranial haemorrhage, seizures, hardware complications and other complications. RESULTS: 130 patients were treated (62 women, 68 men; average age 62 (36-74) years). The average duration of disease from the time of diagnosis to operation was 15.3 years (4-28 years) and the mean follow-up was of 37 months (3-93 months). One hundred and twenty four patients were implanted bilaterally and 6 unilaterally. 62% did not present any complications, 30% had one complication, and 8% more than one complication. Aborted procedures amounted to 5.14% of all procedures, misplaced leads 2.2%, intracranial haemorrhage 3.3%, seizures 4.7%, hardware complications 1.8% and other complications 5.1%. CONCLUSION: Deep brain stimulation surgery is an effective and safe method to treat Parkinson's disease with a low incidence of permanent adverse events.

Prospects for homologous recombination in human gene therapy.

The ideal approach to gene therapy of hereditary diseases or gene correction therapy is considered. The advantages, disadvantages and limits of gene targeting by homologous recombination are discussed with regard to its possible application in gene correction therapy and in comparison with retroviral-mediated gene complementation therapy.

Presence of sterol-binding sites in the cytosol of French-bean (Phaseolus vulgaris) roots.

Vitamin D3 (cholecalciferol) and stigmasterol have been shown to stimulate Ca2+ uptake and to induce calmodulin synthesis in cultured French-bean (Phaseolus vulgaris) roots. In addition, the appearance of calmodulin in the cultures in response to vitamin D3 could be prevented by RNA-synthesis inhibitors. To investigate the possibility that the sterols affect root DNA transcription through a receptor-mediated mechanism, the existence of sterol-binding sites in P. vulgaris roots was investigated. Specific binding of [3H]vitamin D3 could be demonstrated with intact tissue and the cytosolic fraction obtained therefrom. Equilibrium in the binding reaction with cytosol was attained after 4 h of incubation at 0 degrees C. The [3H]vitamin D3 was reversibly bound, since it could be displaced by an excess of unlabelled sterol. An equilibrium binding constant (KD) of (3.48 +/- 0.09) x 10(-9) M and a maximum binding-site concentration (nmax) of 32 +/- 2.54 (3) pmol/mg of protein could be calculated by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672] analysis. In addition to vitamin D3, stigmasterol and sitosterol were effectively able to compete with [3H]vitamin D3 for binding to root cytosol. Cortisol, oestradiol and progesterone displaced bound labelled vitamin D3 to a lesser extent, whereas 5 beta-dihydrotestosterone, lanosterol and diosgenin were ineffective. The affinity and specificity of the root sterol-binding sites are in agreement with the characteristics of tissue responses to the sterols (Ca2+ uptake and calmodulin synthesis).

Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family.

The thrombospondin and collagen type I receptor CD36 is a plasma membrane glycoprotein present in a restricted number of cell types. By contrast, rat lysosomal integral membrane protein II (LIMPII) is expressed on the membrane of lysosomes in all cell types so far examined. Nevertheless, both belong to the same gene family based on alignment of their respective amino acid sequences. To explore the existence of other related members, we have used the polymerase chain reaction with primers derived from highly conserved amino acid regions between CD36 and rat LIMPII. A human cDNA corresponding to a novel member of this family has been identified and isolated. This new member has been designated as CD36 and LIMPII Analogous-1 (CLA-1). Human CLA-1 cDNA predicts a protein 409 amino acids long with a 20% amino acid identity with CD36 and rat LIMPII. Further studies revealed that the sequenced cDNA clone may result by alternative splicing from a longer mRNA form having an insertion of 300 nucleotides located 126 nucleotides downstream from the initiation codon of cloned CLA-1. This form would encode a protein 509 amino acids long, whose sequence matches without any long gap to amino acid sequences of CD36 and rat LIMPII. Northern blot analysis indicates that CLA-1 is widely expressed although its mRNA steady state levels vary considerably among the analyzed cell types. Transient transfection experiments of a CD36-CLA-1 chimera, constructed by replacing the carboxyl cytoplasmic tail of CD36 for the carboxyl cytoplasmic tail of CLA-1, suggest that native CLA-1 protein is found on the plasma membrane.