Cycloaddition between nitrogen-doped graphene (6π-component) and benzene (4π-component): a theoretical approach using density functional theory with vdW-DF correction.

The interaction between nitrogen-doped graphene defects (N3V1 and N4V2 pyridinic, and N3V1 and N3V3 pyrrolic) and benzene have been investigated by applying density functional theory (DFT), together with the vdW-DF correction. We discovered that only the N3V3 pyrrolic defect is a reactive site (6π-component), forming a cycloadduct with benzene (4π-component) that has energy barriers below 154.38 kJ mol-1 (1.60 eV). The conduction and valence bands (HOMO and LUMO) for N3V3 form a degenerate pair of orbitals at the gamma point, with the same ionization potential (IP) and electron affinity (EA). Likewise, inspection of the orbital symmetries for both systems confirms that these must undergo concerted reactions based on the Woodward and Hoffmann principles of orbital symmetry, with the appropriate orbital occupancies. This is the first time that substitutionally doped graphene has been demonstrated to participate as a 6π-component for cycloaddition reactions with benzene.

Study of the interplay between N-graphene defects and small Pd clusters for enhanced hydrogen storage via a spill-over mechanism.

The hydrogen spill-over mechanism was studied by applying Density Functional Theory. We used small palladium clusters to act as the catalyst supported on the substrate (comprised of pyridinic and pyrrolic nitrogen doped graphene), in order to study hydrogen dissociation, migration and diffusion. Charge transfer and strong binding between the catalyst and the substrate lead to dissociated states of H2 and prevent clusters from detaching and coalescing. In dissociated cases of H2 on Pd clusters, energy barriers below 0.6 eV were found. Likewise, concerning hydrogen migration from the catalyst to the substrate, energy barrier values of 0.8 eV (pyridinic defect) and 0.5 eV (pyrrolic defect) were apparent in the case of the Pd4 cluster at full hydrogen saturation. This indicates that hydrogen dissociation and migration may occur spontaneously at room temperature. This result shows that the interaction between the defects and the small metal clusters may explain the role that defects play in hydrogen migration from the catalyst to the substrate. Subsequently, it was found that thermal desorption does not limit chemisorbed hydrogen diffusion on the substrate. This work may thus help to determine experimental strategies with the capacity to enhance hydrogen storage.

Changes of the nucleolus architecture in absence of the nuclear factor CTCF.

CTCF is a multifunctional nuclear factor involved in many cellular processes like gene regulation, chromatin insulation and genomic organization. Recently, CTCF has been shown to be involved in the transcriptional regulation of ribosomal genes and nucleolar organization in Drosophila cells and different murine cell types, including embryonic stem cells. Moreover, it has been suggested that CTCF could be associated to the nucleolus of human erythroleukemic K562 cells. In the present work, we took advantage of efficient small hairpin RNA interference against human CTCF to analyze nucleolar organization in HeLa cells. We have found that key components of the nucleolar architecture are altered. As a consequence of such alterations, an upregulation of ribosomal gene transcription was observed. We propose that CTCF contributes to the structural organization of the nucleolus and, through epigenetic mechanisms, to the regulation of the ribosomal gene expression.

Upper gastrointestinal bleeding: risk factors for mortality in two urban centres in Latin America.

OBJECTIVE: To describe the experience with upper gastrointestinal bleeding in two major Latin American hospitals; presenting its main causes, treatment, and prognosis, while exploring some risk factors associated with death. DESIGN: Prospective cohort study. PATIENTS AND METHODS: Four hundred and sixty four patients were admitted into any of the 2 hospitals and were at least 15 years of age. Some variables demographics, clinics and treatment were studied. The association between those variables and the death were explored. RESULTS: Mean age was 57.9 years; the men:women ratio was 1.4:1. Three hundred and fifty nine patients (77.3%) presented as outpatients and 105 patients (22.6%) were inpatients presenting with UGIB. 71.6% of patients had an upper GI endoscopy within 24 hours. The main causes of bleeding were peptic ulcer (190 patients, 41%), erosive disease (162 patients, 34.9%) and variceal bleeding (47 patients, 10.1%). Forty four patients died (9.5%). Bleeding as an inpatient has a higher mortality risk than does bleeding as an outpatient (RR 2.4 IC 95% 1.2-4.6). An increasing number of comorbidities such as those described in the Rockall Score are also associated with a higher risk of dying (RR 2.5 IC 95% 1.1-5.4). CONCLUSION: UGIB as an inpatient and the presence of comorbidities should alert the clinician in identifying patients at higher risk of a fatal outcome, these patients should have a more aggressive management and be entitled to an early intervention.

Chromatin structure contribution to the synaptonemal complex formation.

Meiosis is a key cellular and molecular process for sexual reproduction contributing to the genetic variability of organisms. This process takes place after DNA replication and consists in a double cellular division, giving rise to four haploid daughter cells or gametes. Meiotic recombination between homologous chromosomes, in the meiotic prophase I, is mediated by a tripartite structure named Synaptonemal Complex (SC). The SC is a peptidic scaffold in which the chromatin of homologous chromosomes is organized during the pachytene stage, holding chromosomes together until the meiotic recombination and genetic exchange have taken place. The role of chromatin structure in formation of the SC and the meiotic recombination at meiotic prophase I remain largely unknown. In this review we address the epigenome contribution to the SC formation at meiotic prophase I, with particular attention on the chromatin structure modifications occurring during the sub-stages of meiotic prophase I.

[Primary central nervous system lymphoma: clinical experience in a neurological center]. / Linfoma primario del sistema nervioso central: experiencia clinica en un centro neurologico.

INTRODUCTION: Primary lymphoma of the central nervous system is a variety of non-Hodgkin's lymphoma that accounts for 4-5% of intracranial tumours and 5% of all lymphomas. It has its origin in the brain, the eyes, the leptomeninges and the spinal cord with no systemic evidence of lymphomatoid activity; the subtype of lymphoma is predominantly of B-type cells. PATIENTS AND METHODS: We conducted a descriptive study of the patients diagnosed with primary brain lymphoma who were attended to at third-level centres in Mexico between the years 1980 and 2016. Patients who had been screened for systemic lymphoma were included. The results were analysed by means of simple frequencies, and disease-free and overall survival time was analysed by Kaplan-Meier curves; the differences among curves were analysed by means of log rank. RESULTS: Of a total of 215 patients, there were only 74 cases. By sex, 45% were females and 55% were males. Regarding age, 36.7% were over 60 years old. The most frequent clinical manifestations were motor loss (60%) and cognitive disorders (52%). Most patients received some form of chemotherapy (89%). The only significant factor for radiological response and clinical prognosis was the combined use of radiochemotherapy (p = 0.04493). CONCLUSION: Lymphoma is a tumorous condition with a high clinicoradiological response to treatment, although the response is not long-lasting. Its early identification and multidisciplinary management are essential for a more favourable prognosis in these patients.

TITLE: Linfoma primario del sistema nervioso central: experiencia clinica en un centro neurologico.Introduccion. El linfoma primario del sistema nervioso central es una variedad de linfoma no Hodgkin que representa el 4-5% de los tumores intracraneales y el 5% de todos los linfomas. Se origina en el encefalo, los ojos, la leptomeninge y la medula espinal sin evidencia sistemica de actividad linfomatoide; el subtipo de linfoma mayoritariamente es de celulas de tipo B. Pacientes y metodos. Estudio descriptivo de los pacientes diagnosticados con linfoma cerebral primario que fueron atendidos en centros de tercer nivel en Mexico entre los años 1980 y 2016. Se incluyo a los pacientes que contaran con cribado para busqueda de linfoma sistemico. Los resultados se analizaron mediante frecuencias simples; en el caso del tiempo libre de enfermedad y supervivencia global, mediante curvas de Kaplan-Meier, y las diferencias entre curvas, mediante log rank. Resultados. En un total de 215 pacientes solo hubo 74 casos. El 45% fueron mujeres y el 55%, hombres. El 36,7% eran mayores de 60 años. Las manifestaciones clinicas mas frecuentes fueron deficit motor (60%) y alteraciones cognitivas (52%). La mayoria recibio alguna forma de quimioterapia (89%). El unico factor significativo para respuesta radiologica y pronostico clinico era el uso combinado de radioquimioterapia (p = 0,04493). Conclusion. El linfoma representa una patologia tumoral con alta respuesta clinicorradiologica al tratamiento, aunque la respuesta no es duradera. Es fundamental su identificacion temprana y el tratamiento multidisciplinario para el mejor pronostico de estos pacientes.

Radiotherapy plus temozolomide or PCV in patients with anaplastic oligodendroglioma 1p19q codeleted. / Radioterapia mas temozolomida o PCV en pacientes con oligodendroglioma anaplasico con codelecion 1p19q.

INTRODUCTION: Radiotherapy with procarbazine, lomustine, and vincristine (PCV) improves overall survival in patients with anaplastic oligodendroglioma 1p19q codeleted. PATIENTS AND METHODS: This retrospective analysis investigated outcomes in patients with anaplastic oligodendroglioma 1p19q codeleted compared two different protocols (radiotherapy plus temozolomide or PCV). The primary end points were overall survival and progression-free survival. Secondary endpoint was the radiological response. RESULTS: A total of 48 patients were included. Mean age was 43 years (range: 19-66 years), 26 were male (54.1%). Twenty-one patients received PCV and 27 temozolomide. The baseline characteristics were not difference between the groups. The progression-free survival and overall survival in the PCV group were 7.2 and 10.6 years respectively and temozolomide were 6.1 and 9.2 years, both statistically significant. The radiological response was present in 80.9% in PCV arm and 70.2% in temozolomide arm there was not statistical differences. The multivariate Cox model showed only the significant parameters the use of PCV protocol. The toxicity grade 3 or 4 was present in 42.8% in PCV arm and 11.1% in temozolomide arm. CONCLUSIONS: The most common strategy in the Latin America community is the substitution of the PCV for temozolomide. This retrospective study showed superior efficacy of PCV than temozolomide. The Latin American community effort must be made to be able to have the drugs to available for using as a first line of treatment.

TITLE: Radioterapia mas temozolomida o PCV en pacientes con oligodendroglioma anaplasico con codelecion 1p19q.Introduccion. La radioterapia con procarbacina, lomustina y vincristina (PCV) mejora la supervivencia global en pacientes con oligodendroglioma anaplasico con codelecion 1p19q, pero no esta disponible en America Latina. Pacientes y metodos. Analisis retrospectivo comparando dos protocolos diferentes, radioterapia mas temozolomida o PCV, en pacientes con oligodendroglioma anaplasico con codelecion 1p19q. Los objetivos primarios fueron la supervivencia global y la supervivencia libre de progresion, y el objetivo secundario, la respuesta radiologica. Resultados. Se incluyo a 48 pacientes, 26 de ellos varones (54,1%), con una edad media de 43 años (rango: 19-66 años). Veintiun pacientes recibieron PCV, y 27, temozolomida. Las caracteristicas iniciales no tuvieron diferencias entre los grupos. La supervivencia libre de progresion y la supervivencia global en el grupo con PCV fueron de 7,2 y 10,6 años, y en el grupo de temozolomida, de 6,1 y 9,2 años, respectivamente, unos resultados estadisticamente significativos. Hubo respuesta radiologica en el 80,9% en el brazo de PCV y el 70,2% en el brazo de temozolomida. El analisis multivariado de Cox mostro como unico parametro significativo el uso del protocolo PCV. El grado de toxicidad 3-4 estuvo presente en el 42,8% en el brazo de PCV y en el 11,1% en el brazo de temozolomida. Conclusiones. La estrategia mas comun en America Latina es la sustitucion de PCV por temozolomida. Este estudio retrospectivo mostro una eficacia superior de PCV que de la temozolomida. La diferencia obliga a la comunidad latinoamericana a hacer un esfuerzo colectivo para poder tener acceso a los medicamentos para su uso como primera linea de tratamiento.

Changes to the dissociation barrier of H2 due to buckling induced by a chemisorbed hydrogen on a doped graphene surface.

An effectiveway of enhancing hydrogen storage on adsorbent materials can be induced by the hydrogen spill-over mechanism, although to date there is no general consensus which satisfactorily explains the mechanism. In this work, a possible reaction path to explain hydrogen adsorption is shown. Density-functional calculations were used to study the dissociation of molecular hydrogen near to a stressed region, as a consequence of chemisorbed hydrogen at the graphene-nitrogen surface. We found that as a result of the buckling induced by the chemisorbed hydrogen, the dissociation barrier of molecular hydrogen diminished by 0.84 eV. The chemisorbed hydrogen is the final state in the spill-over mechanism on a graphene-nitrogen decorated with palladium clusters. This effect helps to create hydrogen nanoislands that may change the diffusion and detrapping of H. An electronic structure analysis suggests that these systems occasionally present metallic or semiconductor behavior. Graphical Abstract Hydrogen dissociation and adsorption process via buckling defect.

Prodos is a conserved transcriptional regulator that interacts with dTAF(II)16 in Drosophila melanogaster.

The transcription factor TFIID is a multiprotein complex that includes the TATA box binding protein (TBP) and a number of associated factors, TAF(II). Prodos (PDS) is a conserved protein that exhibits a histone fold domain (HFD). In yeast two-hybrid tests using PDS as bait, we cloned the Drosophila TAF(II), dTAF(II)16, as a specific PDS target. dTAF(II)16 is closely related to human TAF(II)30 and to another recently discovered Drosophila TAF, dTAF(II)24. PDS and dTAF(II)24 do not interact, however, thus establishing a functional difference between these dTAFs. The PDS-dTAF(II)16 interaction is mediated by the HFD motif in PDS and the N terminus in dTAF(II)16, as indicated by yeast two-hybrid assays with protein fragments. Luciferase-reported transcription tests in transfected cells show that PDS or an HFD-containing fragment activates transcription only with the help of dTAF(II)16 and TBP. Consistent with this, the eye phenotype of flies expressing a sev-Ras1 construct is modulated by PDS and dTAF(II)16 in a gene dosage-dependent manner. Finally, we show that PDS function is required for cell viability in somatic mosaics. These findings indicate that PDS is a novel transcriptional coactivator that associates with a member of the general transcription factor TFIID.

BIOLOGICAL PROPERTIES AND CHEMICAL COMPOSITION OF JATROPHA NEOPAUCIFLORA PAX.

BACKGROUND: Ethnopharmacological relevance. Jatropha neopauciflora (Pax) is an endemic species of the Tehuacan- Cuicatlan Valley, Mexico. This species has long been used as a remedy to alleviate illnesses of bacterial, fungal and viral origin. Aim of the study. Experimentally test the traditional use of Jatropha neopauciflora in Mexican traditional medicine. MATERIALS AND METHODS: The methanol extract (MeOH1), of Jatropha neopauciflora (Euphorbiaceae) was obtained by maceration. Next, the methanol (MeOH2) and hexane (H) fractions were obtained. The essential oil was obtained by hydro- distillation. The extract, fractions and essential oil were analyzed by GC-MS. The antimicrobial activity was measured by the disc diffusion agar and radial inhibition growth methods. RESULTS: The extract and fractions showed antibacterial activity against eleven strains (five Gram-positive and six Gram- negative) and a bacteriostatic effect in the survival curves for Staphylococcus aureus and Vibrio cholerae. The extract and fractions were also shown to have antifungal activity, particularly against Trichophyton mentagrophytes (CF50 = MeOH1: 1.07 mg/mL, MeOH2: 1.32 mg/mL and H: 1.08 mg/mL). The antioxidant activity of MeOH1 (68.6 µg/mL) was higher than for MeOH2 (108.1 µg/mL). The main compounds of the essential oil were ß-pinene, 1,3,8-p-menthatriene, ledene, m- menthane, linalyl acetate and 3-carene. The main compounds of MeOH1 were ß-sitosterol, lupeol and pyrogallol; the main compounds of MeOH2 were ß-sitosterol, spathulenol, coniferyl alcohol and lupeol; and the main compounds of H were ß-sitostenone, γ-sitosterol and stigmasterol. CONCLUSIONS: This study indicates that Jatropha neopauciflora is a potential antibacterial and antifungal agent.

Antimicrobial and anti-inflammatory activities, wound-healing effectiveness and chemical characterization of the latex of Jatropha neopauciflora Pax.

ETHNOPHARMACOLOGICAL RELEVANCE: Jatropha neopauciflora Pax is an endemic species to Mexico, and its latex is used in traditional medicine to treat mouth infections when there are loose teeth and to heal wounds. In this research, we evaluated the antimicrobial activity, wound healing efficacy and chemical characterization of J. neopauciflora latex in a murine model. MATERIALS AND METHODS: The antibacterial activity was determined using Gram positive and negative strains, the antifungal activity was determined using yeast and filamentous fungi, and the wound healing efficacy of the latex was determined using the tensiometric method. The anti-inflammatory activity was evaluated using the plantar oedema model in rats, administering the latex orally and topically. Cytotoxic activity was determined in vitro in two different cell lines. Antioxidant capacity, total phenolics, total flavonoids, reducing carbohydrates and latex proteins were quantified. The latex analysis was performed by High Performance Liquid Chromatography (HPLC). Finally, molecular exclusion chromatography was performed. RESULTS: The latex demonstrated antibacterial activity. The most sensitive strains were Gram positive bacteria, particularly S. aureus (MIC=2mg/mL), and the latex had bacteriostatic activity. The latex did not show antifungal activity. The latex demonstrated a wound-healing efficacy, even the positive control (Recoveron). The orally administered latex demonstrated the best anti-inflammatory activity and was not toxic to either of the 2 cell lines. The latex had a high antioxidant capacity (SA50=5.4µg/mL), directly related to the total phenolic (6.9mg GAE/mL) and flavonoid (12.53µg QE/mL) concentration. The carbohydrate concentration was 18.52µg/mL, and fructose was the most abundantly expressed carbohydrate in the latex (14.63µg/mL, 79.03%). Additionally, the latex contained proteins (7.62µg/mL) in its chemical constitution. As secondary metabolites, the HPLC analysis indicated the presence of phenols and flavonoids. CONCLUSIONS: The J. neopauciflora latex promotes the wound healing process by avoiding microorganism infections, inhibiting inflammation and acting as an antioxidant.

[Percutaneous coronary intervention in Navarre. Outcomes of a low volume centre]. / Intervencionismo coronario percutáneo en Navarra. Resultados de un centro de bajo volumen intervencionista.

BACKGROUND: Percutaneous coronary intervention (PCI)is currently a basic therapeutic option in patients with coronary artery disease. To carry this out specialists must be trained and accredited. It is known that the number of procedures performed each year influences results. We suggest that some low volume centres may also get good results. METHODS: Prospective analysis of clinical features and immediate results obtained in our centre following PCI performed between 2006 and 2012 and retrospective analysis of overall survival, outcome-free survival and restenosis in patients treated between 2006 and 2009.The clinical features, acute and long-term events (complications,survival and mortality) of our group were compared with other published studies. RESULTS: In our centre the likelihood of complications ina PCI was 9% with an overall mortality of 2%. PCI mortality in stable coronary disease was 0.43% and in acute coronary syndrome 6.25%. Complications at the vascular access site was 1.44% and restenosis at nine months, inpatients undergoing PCI for the first time, was 5.2%. CONCLUSIONS: Although a high interventionist volume has been shown to reduce the rate of complications and improve long-term evolution, some low volume interventional centres can obtain similar results to those of high volume interventional centres.

Amphiphilic and hydrophilic forms of acetylcholinesterase from sheep platelets.

Acetylcholinesterase (AChE, EC 3.1.1.7) was extracted from sheep platelets by successive homogenizations, yielding low-salt soluble (LSS), high-salt soluble (HSS) and detergent-soluble (DS) fractions. These accounted, respectively, for about 30%, 7% and 60% of total AChE activity. Applications of hydrophobic chromatography on phenyl-agarose to three solubilized fractions revealed that hydrophilic forms were almost exclusively located in the LSS fraction ( approximately 27% of total AChE), whereas most amphiphilic forms were present in DS extracts ( approximately 59% of total AChE), the remaining forms being distributed among aqueous soluble fractions. Enzyme molecular forms in the solubilized extracts were identified by centrifugation in 5-20% sucrose gradients containing Triton X-100 or Brij 97 to differentiate between hydrophilic or amphiphilic species. A predominance of hydrophilic dimeric forms ( approximately 22%), with small amounts of hydrophilic monomers (5%) and amphiphilic dimers and monomers (3%), was found in soluble AChE (LSS fraction). Amphiphilic AChE forms extracted in the HSS and DS fractions had a single peak in the sedimentation profiles with sedimentation coefficients of about 6S in gradients with Triton X-100; these were slightly shifted in the presence of Brij 97. After treatment with dithiothreitol, this molecular form solubilized in DS was converted to another molecular form with a lower sedimentation coefficient. Our results show that amphiphilic globular dimers are the dominant molecular form in sheep platelet AChE, suggesting a partial conversion of this membrane-bound form into soluble dimers and monomers, mainly with a hydrophilic character, through the action of either endogenous proteases and phospholipases or residual endogenous reducing agents.

Amphiphilic and hydrophilic nature of sheep and human platelet phosphotyrosine phosphatase forms.

To date, although at least 75 different PTPases (protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) have been identified, those detected in platelets are rather scarce. Based on previous results from our laboratory, we investigated the existence of new PTPases in platelets. Triton X-114 phase partitioning of Triton X-100-solubilized human and sheep platelet membranes allowed PTPase to be recovered in the detergent-rich (40-35%, respectively) and -poor phases (60-65%, respectively). Sedimentation analyses of both phases from the sheep species revealed hydrophilic 6S and 3.7S, and amphiphilic 7.5S and 10.3S PTPase forms. Sedimentation analyses of human platelet membrane-associated or cytosolic PTPase revealed hydrophilic 6.7S and 4.3S, and amphiphilic 5.5S and 10.8S forms, or hydrophilic 4S, 5.9S and 6.9S forms, respectively. Western blot analysis using monoclonal antibodies (MoAb) against human PTP1B, PTP1C, PTP1D and RPTPalpha (mouse anti-human PTPase MoAbs) showed that RPTPalpha was not present in platelets and that the PTP1C type and PTP1D type (but probably not the PTP1B type) were expressed in sheep species. Immunoblots also revealed that all PTPases detected were mainly membrane-associated, with similar percentages of cellular distribution in both species. All PTPases were mainly recovered in the detergent-poor phases from the Triton X-114 phase partitioning, although PTP1D from human species was also significantly present (30%) in the detergent-rich phase. Additionally, all PTPases sedimented within the same PTPase peak in sucrose gradients (sedimentation coefficients around 4S). These findings indicate that amphiphilic and hydrophilic PTPases different from PTP1B, PTP1C, PTP1D or RPTPalpha, with higher sedimentation coefficients and with higher activity when O-phosphotyrosine or a synthetic peptide phosphorylated on tyrosine were used as substrates, are present in platelets.

A genetic approach to detect muscle protein interactions in vivo.

An organism is required to identify biologically relevant protein interactions. We propose Drosophila and its indirect flight muscles as a suitable experimental system for genetic screenings for muscle protein interactions. The first attempt focused on troponin I (TnI) in view of the key role in thin filament regulation that this protein performs. Suppressors of a defined Tn I allele have been isolated as mutations in the heavy chain of myosin (MhC). This unsuspected functional interaction between TnI and MhC serves to illustrate one of the benefits of the approach. Four of the suppressors identified to date reside in the MhC head, around the actin-binding site and near the lips of the pocket where ATP is hydrolyzed. Two other suppressors correspond to a second site mutation in TnI and a mutation in the conserved region of Tropomyosin II (TmII), respectively. All the identified suppressors are mutations in constituents of the sarcomere, and most of them are structurally similar to human mutations causing familial hypertrophic cardiomyopathy (FHC). At least seven sarcomere proteins can lead to FHC and, consequently, the disease is heterogeneous and difficult to diagnose. In addition, putative natural suppressors may help obscure the origin of FHC. The genetic procedure, used here for muscle proteins, could help diagnose FHC and other myopathies, and extend to proteins of clinical interest in other tissues, including the nervous and circulatory systems.

His1069Gln and six novel Wilson disease mutations: analysis of relevance for early diagnosis and phenotype.

In the present study we examined 33 German and 10 Cuban unrelated Wilson disease (WND) index patients and their relatives. The common His1069Gln mutation accounted for 42% of all WND chromosomes in the German series and the haplotype C was found to be highly predictive for this mutation. Six WND gene mutations have not been described previously and involved a splice site at intron 18 (3903 + del1G), a termination codon in the copper-binding region of exon 2 (Cys271X), and missense mutations in transmembrane region 2 (Gly710Ala), in transmembrane region 3 (Tyr741Cys), in the DKTGT motif (Thr1031Ile) and in the ATP loop region (Gly1176Arg). In 15 German WND index patients and three sibs both WND mutations could be determined and a genotype-phenotype correlation was attempted. Patients homozygous for the His1069Gln mutation showed almost the complete range of clinical presentations, and thus in our study this mutation is not associated with a late, neurological presentation.

Oxidative inactivation of human and sheep platelet membrane-associated phosphotyrosine phosphatase activity.

Incubation of human or sheep platelet crude membranes with xanthine oxidase/hypoxanthine in the presence of Fe2+/ADP inactivated phosphotyrosine phosphatase (PTPase, protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity in a time-dependent manner, this inhibition being significant within 5 min of treatment. The dynamics of protein thiols differed depending on the platelet species, but in any case decreases in protein thiols were only visible 20-45 min after the start of the treatment. The inhibition of PTPase activity in general showed good a correlation with the production of thiobarbituric acid-reactive substances (TBARS). The results with several antioxidants suggest that the inhibition of PTPase activity is related to the generation of alkoxyl and/or peroxyl radicals. Furthermore, the formation of fluorescent products and changes in amino groups were observed only after long incubation times with the oxidizing agents, these fluorescent products and the residual enzyme activity remaining in the membrane fraction. Treatment of platelet membranes with trans-2-nonenal and n-heptaldehyde, but not with acetaldehyde, also inhibited membrane-associated PTPase activity. However, the amount of protein thiols was reduced only by treatment with trans-2-nonenal. Fluorescence product formation was always higher with trans-2-nonenal, these products being mainly located in the protein fraction. The results with aldehydes suggest that secondary degraded products of lipid hydroperoxides affect PTPase activity. Kinetic studies of PTPase activity indicated that with all treatments enzyme inhibition is mainly due to a decrease in the Vmax value. The results of fluorescence anisotropy measurements of labeled platelet membranes did not support the notion of a contribution of the lipid organization to peroxidation-mediated PTPase inhibition. All the above results indicate that platelet membrane-associated PTPase inhibition due to treatment with xanthine oxidase/ hypoxanthine in the presence of Fe2+/ADP is a very complex, time-dependent process, and that it is probably related, at least after long periods of peroxidation, to changes in protein thiols and amino groups. We predict that the sensitivity of PTPase to lipid peroxidation must be physiologically relevant because of the increasing importance of tyrosine phosphorylation in signal transduction, in general, and in platelet activation and aggregation in particular.

Vesiculation and changes in fluidity and lipid composition of platelet membranes after storage of sheep platelets in plasma or Seto solution.

To relate the improvement of platelet storage in synthetic media with possible structural changes, we conducted serial studies on the membranes of platelets and microparticles shed during platelet storage for up to 5 days at 4 degrees C either in plasma or in Seto solution. Spontaneous microparticle formation proceeded linearly for up to 2 days in both storage media, although the processes seemed to be different because microparticles from Seto solution had a higher lipid/protein ratio than those released in plasma. Microparticles were heterogeneous structures showing beta-N-acetylhexosaminidase, glucose-6-phosphatase and succinate dehydrogenase activities. After 2-5 days of storage, microparticles contained 60% of total cellular acetylcholinesterase (AChE), were doubly enriched in cholesterol. and showed identical phospholipid profiles but with a decrease in the lipid unsaturation index with respect to fresh platelets. Fluorescence anisotropy studies pointed to a remarkable increase in the deep lipid core fluidity of microparticles during storage of platelets in plasma. With respect to platelets, only those stored in plasma showed significant changes in lipid contents, with a 3-fold decrease in the phospholipid to protein ratio, a decrease in phosphatidylethanolamine (PE) levels and a parallel increase in phosphatidylcholine (PC) percentages in their phospholipid profile, together with a significant reduction in the lipid unsaturation index after 1 day of storage. The fluidity of the negatively charged surface of the platelet membranes decreased in platelets stored for 5 days in both media, whereas the fluidity of the membrane deep core was only increased in platelets stored in plasma. These findings suggest that Seto solution permits better storage of platelets for 5 days than plasma and support the notion that lipid peroxidation could play an important role in the structural changes observed.

Preservation of acetylcholine muscarinic M2 receptor G-protein interactions in the neocortex of patients with Alzheimer's disease.

The efficacy of acetylcholine muscarinic M2 receptor-G protein coupling was investigated in Alzheimer's disease and control neocortical membranes by measuring the effects of MgCl2 and 5'-guanylylimidodiphosphate (Gpp[NH]p) on high-affinity [3H]oxotremorine-M ([3H]OXO-M) binding. MgCl2 gave similar enhancements of [3H]OXO-M binding in Alzheimer's disease and control occipital cortex. In contrast, MgCl2 enhanced [3H]OXO-M binding was significantly higher in Alzheimer's disease superior temporal cortex, compared to controls. MgCl2 enhanced [3H]OXO-M binding in both the occipital and temporal cortices of the Alzheimer's disease cases was reversed to control levels by Gpp[NH]p. It is concluded that the number of high-affinity muscarinic M2 sites is increased in Alzheimer's disease superior temporal, but not occipital, cortex and that M2 sites in both regions maintain an efficient G-protein coupling.

The effect of GTP on the aluminum fluoride- and forskolin-activated adenylyl cyclase from human embryonic kidney 293 cells.

GTP has been shown to inhibit AlF4(-)-stimulated, and to activate forskolin-stimulated adenylyl cyclase activity in the presence of Mg2+ in cell membranes from human embryonic kidney 293 cells. The maximal inhibitory response of AlF4(-)-stimulated adenylyl cyclase activity by GTP was not dependent on the concentration of Mg2+, but was so in the case of forskolin-activated activity at all forskolin concentrations assayed. Mn2+ ions stimulated AlF4(-)- or forskolin-activated adenylyl cyclase activity to a greater extent than Mg2+. The inhibition of AlF4(-)-stimulated cyclase by GTP was still observed with Mn2+, but the activation of forskolin-stimulated cyclase by GTP was not. When assayed together, Mn2+ and Mg2+ showed non-additive behaviours with respect to the amount of cyclic AMP formed after AlF4(-)-stimulation of adenylyl cyclase. The temperature dependence of the activation of adenylyl cyclase by forskolin, AlF4- or under basal conditions was observed to be somehow different in the presence of Mn2+ than in the presence of Mg2+ ions. Cholera toxin treatment produced a markedly increased cyclase activity, specially when assayed with AlF4-. In the case of forskolin-activated adenylyl cyclase, UTP and CTP were unable to reproduce the cyclase activation detected with GTP. However, in the case of AlF4(-)-stimulated adenylyl cyclase, UTP was as good as GTP at inhibiting cyclase activity, and CTP virtually eliminated the activation of the cyclase with AlF4-.