Synergistic activation of AMPK prevents from polyglutamine-induced toxicity in Caenorhabditis elegans.

Expression of abnormally long polyglutamine (polyQ) tracks is the source of a range of dominant neurodegenerative diseases, such as Huntington disease. Currently, there is no treatment for this devastating disease, although some chemicals, e.g., metformin, have been proposed as therapeutic solutions. In this work, we show that metformin, together with salicylate, can synergistically reduce the number of aggregates produced after polyQ expression in Caenorhabditis elegans. Moreover, we demonstrate that incubation polyQ-stressed worms with low doses of both chemicals restores neuronal functionality. Both substances are pleitotropic and may activate a range of different targets. However, we demonstrate in this report that the beneficial effect induced by the combination of these drugs depends entirely on the catalytic action of AMPK, since loss of function mutants of aak-2/AMPKα2 do not respond to the treatment. To further investigate the mechanism of the synergetic activity of metformin/salicylate, we used CRISPR to generate mutant alleles of the scaffolding subunit of AMPK, aakb-1/AMPKß1. In addition, we used an RNAi strategy to silence the expression of the second AMPKß subunit in worms, namely aakb-2/AMPKß2. In this work, we demonstrated that both regulatory subunits of AMPK are modulators of protein homeostasis. Interestingly, only aakb-2/AMPKß2 is required for the synergistic action of metformin/salicylate to reduce polyQ aggregation. Finally, we showed that autophagy acts downstream of metformin/salicylate-related AMPK activation to promote healthy protein homeostasis in worms.

Nonsteroidal anti-inflammatory drugs enhance IgE-mediated activation of human basophils in patients with food anaphylaxis dependent on and independent of nonsteroidal anti-inflammatory drugs.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) act as cofactors worsening the allergic reactions induced by food allergens. AIM: The aim of this study was to evaluate the effect of both lysine acetylsalicylate (L-ASA) (non-selective cyclooxygenase (COX) inhibitor) and valdecoxib (selective COX-2 inhibitor) in basophils activated by peach lipid transfer protein (Pru p 3) in patients with food-dependent NSAID-induced anaphylaxis (FDNIA). METHODS: Twenty Pru p 3-allergic patients with FDNIA group, eleven peach anaphylaxis not exacerbated by NSAIDs (no-NSAID group) and 5 healthy volunteers were recruited. Basophil activation (BA) was measured as expression of CD63 (Flow(2) CAST(™) ; Bühlmann(®) ), after stimulation with Pru p 3, both alone and in combination with L-ASA (1.13, 3.38 and 6.78 mm) or valdecoxib (0.87, 7.8 and 31.25 µm). RESULTS: Basophils from no-NSAID group were significantly more reactive and sensitive to Pru p 3 than those from the FDNIA group. In both groups, an increase in BA was observed when basophils were exposed to Pru p 3 and L-ASA. In the FDNIA group, valdecoxib partially terminates the BA induced by Pru p 3, whereas in the no-NSAID group, a dual effect was observed depending on the concentration tested. CONCLUSIONS: This study indicates that subjects with food-induced anaphylaxis differ from FDNIA subjects in the higher reactivity and sensitivity of their basophils to allergen challenge. We have shown a direct effect of NSAIDs on basophils using a human model of FDNIA. Our results also suggest that selective COX2 inhibitors might be a safe alternative. BA test may be a useful tool in the study of the pathogenic mechanism of the cofactor phenomenon.

Asymptomatic LTP sensitisation is common in plant-food allergic children from the Northeast of Spain.

BACKGROUND: The sensitisation profile at molecular level in plant-food allergy is complex. Several allergens may be involved, with different potential for severe reactions. lipid transfer proteins (LTP) are considered the most relevant plant-food allergens in adults in Mediterranean countries, but less is known in children. AIM: To describe the clinical pattern and sensitisation profile of children with plant-food allergy and LTP sensitisation from Northeast Spain. METHODS: Children with history of immediate reaction to plant-food(s), positive skin-prick-test to the culprit plant-food(s) and specific-IgE to plant-food LTPs were analysed. RESULTS: 130 children were included. 69.2% (90/130) had reacted to ≥2 taxonomically unrelated plant-foods. Peach, walnut, hazelnut and peanut were most frequently involved. Reactions severity ranged from anaphylaxis (45.4%, 59/130) to oral symptoms only. Sensitisation to a particular plant-food LTP not always caused clinical symptoms with that plant-food; 69% (40/58) and 63% (17/27) of peach- and walnut-tolerant subjects had positive rPru p 3 and nJug r 3 specific IgE, respectively. 65.4% (85/130) of children were also sensitised to storage proteins, which was associated to anaphylaxis and nut allergy. However, 60% of patients without nuts/seeds allergy were sensitised to storage proteins. Specific-IgE levels to LTPs and/or storage proteins were not useful to predict allergy (vs. tolerance) to peach, walnut, peanut or hazelnut. CONCLUSIONS: Sensitisation to LTP and/or storage proteins without clear clinical significance is relatively common. Prospective longitudinal studies are required to evaluate the relevance of these silent sensitisations over time. Caution is required when interpreting the results of molecular-based diagnostic tools in clinical practice.

Prevalence of urinary tract cancer in the Spanish cohort of the IDENTIFY study.

INTRODUCTION: Malignant tumors of the urinary tract are associated with high morbidity and mortality, and their prevalence can vary worldwide. Recently, the IDENTIFY study has published results on the prevalence of urinary tract cancer at a global level. This study evaluates the prevalence of cancer within the Spanish cohort of the IDENTIFY study to determine whether the published results can be extrapolated to our population. PATIENTS AND METHODS: An analysis of the data from the Spanish cohort of patients in the IDENTIFY study was performed. This is a prospective cohort of patients referred to secondary care with suspected cancer, predominantly due to hematuria. Patients were recruited between December 2017 and December 2018. RESULTS: A total of 706 patients from 9 Spanish centers were analyzed. Of these, 277 (39.2%) were diagnosed with cancer: 259 (36.7%) bladder cancer, 10 (1.4%) upper tract urothelial carcinoma, 9 (1.2%) renal cancer and 5 (0.7%) prostate cancer. Increasing age (OR 1.05 (95% CI 1.03-1.06; P < 0.001)), visible hematuria (VH) OR 2.19 (95% CI 1.13-4.24; P = 0.02)) and smoking (ex-smokers: OR 2.11(95% CI 1.30-3.40; P = 0.002); smokers: OR 2.36 (95% CI 1.40-3.95; P = 0.001)) were associated with higher probability of bladder cancer. CONCLUSION: This study highlights the risk of bladder cancer in patients with VH and smoking habits. Bladder cancer presented the highest prevalence; higher than the prevalence reported in previous series and presented in the IDENTIFY study. Future work should evaluate other associated factors that allow us to create cancer prediction models to improve the detection of cancer in our patients.

Comparing the effect of electron beam, beta and ultraviolet C exposure on the luminescence emission of commercial dosimeters.

This paper reports on the luminescence characterization of TLD-100 (LiF: Ti, Mg), TLD-200 (CaF2: Dy), TLD-400 (CaF2: Mn) and GR-200 (LiF: Mg, Cu, P) dosimeters exposed to electro beam, beta and ultraviolet C radiation -UVC-. All of them show high sensitivity to radiation regardless of whether it is ionizing or partially ionizing radiation based on their luminescence properties (cathodoluminescence -CL- or thermoluminescence -TL-). CL emission differs significantly among them in shape and intensity due to their chemical compositions. LiF samples display three maxima at: (i) 300-450 nm linked to intrinsic and structural defects, (ii) a green waveband probably due to F3+ centres or the presence of hydroxyl groups and (iii) the red-infrared emission band associated with F2 centres. However, CL spectra from the CaF2 dosimeters display meaningful differences due to the dopant. TLD-200 is characterized by an emission with four sharp individual peaks in the green-IR spectral region (due to the Dy3+), whilst TLD-400 exhibits a broad maximum peaked at Ì´500 nm (linked to the Mn2+). On the other hand, the variation in the TL glow curves allows to discriminate the TLDs exposed to beta and UVC radiation since they give rise to different chemical-physical processes and that have been studied from the estimation of the kinetic parameters by means of the Computerised Glow Curve Deconvolution (CGCD) method.

Lipid transfer protein syndrome: clinical pattern, cofactor effect and profile of molecular sensitization to plant-foods and pollens.

BACKGROUND: Multiple plant-food sensitizations with a complex pattern of clinical manifestations are a common feature of lipid transfer protein (LTP)-allergic patients. Component-resolved diagnosis permits the diagnosis of the allergen sensitization profile. OBJECTIVE: We sought to clinically characterize and describe the plant-food and pollen molecular sensitization profile in patients with LTP syndrome. METHODS: Forty-five subjects were recruited, after being diagnosed with multiple plant-food allergies sensitized to LTP, but not to any other plant-food allergen, according to the molecular allergen panel tested (Pru p 3 (LTP), Pru p 1 (Bet v 1-like), Pru p 4 (profilin) and those included in a commercial microarray of 103 allergenic components). IgE-mediated food-allergy symptoms and pollinosis were collected. Patients were skin prick tested with a plant-food and pollens panel, and specific IgE to Tri a 14 was evaluated. RESULTS: A heterogeneous group of plant-foods was involved in local and systemic symptoms: oral allergy syndrome (75.6%), urticaria (66.7%), gastrointestinal disorders (55.6%) and anaphylaxis (75.6%), 32.4% of which were cofactor dependent (Non-Steroidal Anti-inflammatory Drugs, exercise). All tested subjects were positive to peach and Pru p 3, Tri a 14 and to some of the LTPs included in the microarray. Pollinosis was diagnosed in 75.6% of subjects, with a broad spectrum of pollen and pollen-allergen sensitization. Plane tree and mugwort were the statistically significant pollens associated with Pru p 3. CONCLUSIONS AND CLINICAL RELEVANCE: Several plant-foods, taxonomically unrelated, independent of peach involvement, are implicated in LTP syndrome. Local symptoms should be evaluated as a risk marker for anaphylaxis because they are frequently associated with cofactor-dependent anaphylaxis. The association of these symptoms with pollinosis, especially plane tree pollinosis, could be part of this syndrome in our area.

Hirano body-rich subtypes of Creutzfeldt-Jakob disease.

BACKGROUND: In definite Creutzfeldt-Jakob disease (CJD), morphological and immunohistochemical patterns are useful to identify molecular subtypes. Severe cerebellar pathology and hippocampal involvement helps to identify VV subtypes. The rare VV1 variant (<1%), more frequent in young individuals, is additionally characterized by the presence of ballooned neurones in affected areas. In 1985, Cartier et al. described a family cluster of three individuals with an ataxic CJD form, showing, in addition to severe cerebellar and hippocampal involvement, the presence of frequent Hirano bodies (HB) in CA1 pyramidal neurones. HB are frequently found in aged individuals with Alzheimer pathology although they are not a specific finding. AIMS AND METHODS: In this study, we evaluated the presence of HB in hippocampi of 54 genetically and molecularly characterized CJD cases, aiming to elucidate whether additional morphological features could be helpful to point to molecular subtypes. RESULTS: We identified nine cases (four VV1, one out of three MV2K, three out of six MV2K+2C and one MV carrying a 96-base pair insertion) with abundant, partly bizarre and clustered HB in CA1 sector, not observed in other subtypes. The presence of HB was independent of hippocampal involvement by the disease itself. CONCLUSIONS: Clusters of abundant HB might be found in rare CJD subtypes such as VV1, MV2K/MV2K+2C and some genetic cases. In addition to histopathological and PrP immunohistochemical deposition patterns, their presence might be a useful additional morphologic feature that could point to the molecular subtype, especially when genetic and/or Western blot analyses are not available.

PICOGEN: five years experience with a genetic counselling program for dementia.

INTRODUCTION: We describe the 5 year experience of a genetic counselling program for familial dementias (the PICOGEN program). METHODS: The neurologist selected the candidates for genetic testing in the screening visit based on family history and phenotype (Alzheimer disease-AD, frontotemporal lobar degeneration-FTLD, or prion disease). Asymptomatic subjects who decided to know their genetic status were evaluated within a structured protocol by the psychiatrist and psychologist prior to entering the program and followed up afterwards. RESULTS: A total of 87 patients from 72 families were candidates for the genetic study, 20 of the 72 families had a family history of autosomal dominant early-onset dementia (ADEOD). A pathogenic mutation was found in 22 patients (8 PSEN1, 1 PSEN2, 1 APP, 4 MAPT, 8 PRNP), 5 of which had not been previously described. All positive cases, except for 1 PSEN1 (12.5%) and 4 PRNP (50%) showed ADEOD. In 3 ADEOD cases (15%) no pathogenic mutation was found. After individual genetic counselling, 24/54 asymptomatic subjects at risk decided to have the pre-symptomatic study, of whom 10 (42%) were carriers of the pathogenic mutation. In the follow up, no major psychiatric complication was observed. CONCLUSIONS: In our series, family history of ADEOD was a sensitive criterion for the detection of pathogenic mutations in AD and FTLD but not in prion diseases. No genetic anomalies were detected in 15% of the ADEOD cases using conventional diagnostic procedures, and 43% of pre-symptomatic subjects at risk who received individual genetic counselling decided to have the study. The pre-symptomatic diagnosis proved to be safe under these conditions.

The cytotoxic T cell response to the male-specific histocompatibility antigen (H-Y) is controlled by two dominant immune response genes, one in the MHC, the other in the Tar alpha-locus.

The genetic control of the cytotoxic T-cell response to the male histocompatibility antigen, H-Y, was analyzed in BALB/cKe(C) and SJL/J(J) which are both nonresponders. However, the (C X J)F1 hybrid is a responder. Therefore, two dominant complementing genes are involved. Analysis of a set of (C X J) recombinant inbred (RI) lines reveals that these two complementing gene products are a restricting element (R) encoded by the H-2 (MHC) locus on chromosome 17 and a subunit of the T-cell receptor (anti-R) encoded by the Tar alpha-locus on chromosome 14. The order and orientation of gene segments within the Tar alpha-locus has also been established relative to the chromosome 14 marker, Es-10. The existence of two RI strains which are recombinant at chromosome 14 has made it possible to determine that this order is Es-10--v alpha-1--v alpha-2--[C alpha--Np-2]--centromere. The implications of these data for the antigen-specific regulation of immune responsiveness are discussed in terms of the dual recognitive-single receptor model.

An N-acetylated natural ligand of human histocompatibility leukocyte antigen (HLA)-B39. Classical major histocompatibility complex class I proteins bind peptides with a blocked NH(2) terminus in vivo.

Sequence-independent interactions involving the free peptidic NH(2) terminus are thought to be an essential feature of peptide binding to classical major histocompatibility complex (MHC) class I proteins. Challenging this paradigm, a natural Nalpha-acetylated ligand of human histocompatibility leukocyte antigen (HLA)-B39 was identified in this study. It matched the NH(2)-terminal sequence of two human helicases, was resistant to aminopeptidase M, and was produced with high yield from a synthetic 30 mer with the sequence of the putative parental protein by the 20S proteasome. This is the first reported natural ligand of classical MHC class I antigens that has a blocked NH(2) terminus.

Molecular genetic analysis of 178 I-Abm12-reactive T cells.

We have studied the genetic diversity of the TCR repertoire to the murine alloantigen I-Abm12 by generating a panel of 178 C57BL/10-derived I-Abm12-reactive T cell hybridomas. The expression of V alpha and V beta gene families was examined in this panel and the frequency of expression of V beta, but not ofV alpha, gene families differed significantly from that observed in a companion panel of random C57BL/10-derived hybridomas. The V beta 5 gene family was expressed significantly less frequently while the V beta 14, V beta 15, and V beta 16 genes were expressed significantly more frequently in the panel of I-Abm12-reactive than in the panel of random hybridomas. The junctional regions (VJ alpha and VDJ beta) of TCR V alpha and V beta genes from selected I-Abm12-specific hybridomas were amplified using the polymerase chain reaction, and directly sequenced. Surprisingly, no conserved J alpha, D beta, J beta, or N region-encoded sequences among these selected I-Abm12-reactive TCRs were identified. Thus, the T cell response to an I-A alloantigen that differs by only three amino acid residues from the I-A molecule of the responding strain is genetically complex but nonrandom. We have estimated that the repertoire to this alloantigen is comprised of at least 37 different TCRs.

Possible association between NOD2 variants and joint surgery in psoriatic arthritis.

BACKGROUND: Psoriatic arthritis (PsA) has been inconsistently associated with common NOD2 gene variants, although some of these studies did not include patient stratification by clinical phenotype. OBJECTIVES: To analyse the association between the three common NOD2 variants (R702W, G908R and L1007fs) and clinical phenotypes of PsA, particularly with surrogate markers of severe joint destruction. PATIENTS AND METHODS: A total of 183 unrelated PsA patients and 187 controls were included. Demographic, clinical, biological and immunological characteristics were collected. Genotypes for the three common NOD2 gene variants were obtained by PCR and direct sequencing. RESULTS: NOD2 variants in PsA patients (7.6%) are just as prevalent as in healthy controls (7.5%). 18.5% of PsA patients carrying at least one NOD2 variant underwent joint surgery compared with 4.5% of those without these variants (p=0.019). Multivariate analysis confirmed this finding (OR 8.82, CI 1.7-46.3). There was no requirement for early surgery in patients carrying the NOD2 variants but there was an increased possibility of requiring surgery at similar times of disease duration. No other association with clinical features and NOD2 status carrier was found. CONCLUSIONS: Common NOD2 gene variants are not associated with PsA, but might increase the risk of undergoing joint replacement surgery, suggesting that this autoinflammatory-associated gene could act as a phenotypic modifier gene in PsA patients by increasing the risk of joint destruction. Given the small number of PsA patients with joint surgery included, we consider our findings a new hypothesis that will need further testing.

Combined type-1 plasminogen activator inhibitor and NOD2/CARD15 genotyping predicts complicated Crohn's disease behaviour.

BACKGROUND: NOD2/CARD15 gene variants have not been universally associated with stricturing behaviour in Crohn's disease. Other behaviour modifying genes could explain these results. AIM: To study the combined influence of NOD2/CARD15 variants and 4G/4G genotype of type-1 plasminogen activator inhibitor (PAI-1) gene on Crohn's disease behaviour. METHODS: One hundred and seventy Crohn's disease patients were studied prospectively, with a mean follow-up of 7+/- 6 years. Disease behaviour was registered by using two criteria: the Vienna classification and a non-hierarchical classification based on the behavioural Vienna categories. RESULTS: In the multivariate analysis for stricturing behaviour according to the Vienna categories, only absence of colonic disease (OR, 4.0; 95% CI: 1.49-11.1; P = 0.006) was an independent predictive factor. However, in the multivariate analysis for stricturing disease applying a non-hierarchical criteria, ileal disease (OR, 4.19; 95% CI: 1.30-13.5; P = 0.01), and carrying both NOD2/CARD15 variants and the 4G/4G PAI-1 genotype (OR, 5.02; 95% CI: 1.44-17.48; P = 0.01) were independent predictive factors. In the multivariate analysis for penetrating behaviour, the 4G/4G PAI-1 (OR, 3.10; 95% CI: 1.54-6.23; P = 0.001) and male sex (OR, 2.44; 95% CI: 1.30-4.60; P = 0.005) were independent predictive factors irrespective of criteria applied. CONCLUSIONS: Combined PAI-1 and NOD2/CARD15 genotyping predict complicated Crohn's disease. Patients with these variants could benefit from early interventions.

Aromatase expression in the human temporal cortex.

The expression of the human cyp19 gene, encoding P450 aromatase, the key enzyme for estrogen biosynthesis, involves alternative splicing of multiple forms of exon I regulated by different promoters. Aromatase expression has been detected in the human cerebral cortex, although the precise cellular distribution and promoter regulation are not fully characterized. We examined the variants of exon I of cyp19 by PCR analysis and the cellular distribution of the enzyme using immunohistochemistry in the human temporal cortex. We detected four different variants of exon I, suggesting a complex regulation of cyp19 in the cerebral cortex. In addition, the enzyme was localized mainly in a large subpopulation of pyramidal neurons and in a subpopulation of astrocytes. However, the majority of GABAergic interneurons identified by their expression of the calcium-binding proteins calbindin, calretinin and parvalbumin, did not display aromatase immunoreactivity. The broad range of potential modulators of the cyp19 gene in the cortex and the widespread expression of the protein in specific neuronal and glial subpopulations suggest that local estrogen formation may play an important role in human cortical function.

No association of the Val66Met polymorphism of brain-derived neurotrophic factor (BDNF) to multiple sclerosis.

Brain-derived neurotrophic factor (BDNF), a neurotrophin produced by neurons and immune cells, promotes neuronal survival and repair during development and after CNS injury. The BDNF-Val66Met polymorphism is functional and induces abnormal intracellular trafficking and decreased BDNF release. Therefore, we investigated the impact of the BDNF-Val66Met polymorphism on the susceptibility and clinical course in a case-control study of 224 multiple sclerosis (MS) Spanish patients and 177 healthy controls. We found no evidence for association to susceptibility or severity of the disease in our population. Moreover, we did not observe, in a subgroup of 12 MS patients, that the methionine substitution at position 66 in the prodomain had negative impact in the capacity to produce BDNF by peripheral blood mononuclear cells (PBMC).

Loss of heterozygosity of p53 gene and p53 protein expression in human colorectal carcinomas.

The p53 gene is a tumor suppressor gene located on chromosome 17p. Deletions of this chromosome and point mutations of p53 have been implicated in the development of colonic neoplasms. We have analyzed the loss of heterozygosity of the human p53 tumor suppressor gene in 40 cases of colorectal carcinoma using two restriction fragment length polymorphisms detected by BglII and AccII restriction enzymes. p53 gene product expression was studied immunohistochemically in 64 colorectal carcinomas, 18 adenomas, and 40 normal colonic mucosae using an anti-human p53 monoclonal antibody (Pab 1801) and the avidin-biotin-peroxidase complex technique. Twelve of the 40 patients (30%) were polymorphic for the p53 gene. In ten of these informative patients (83%), the tumor samples showed the loss of one allele when compared with normal colorectal samples of the same patient. One of the homozygous patients showed a loss of both p53 alleles. p53 immunostaining was observed in 43 of 64 carcinomas (67%) but only in two adenomas (11%). These two positive adenomas showed areas of carcinoma in situ. The normal mucosa was always negative. No relation could be found between p53 immunostaining and the degree of differentiation, the extension of the tumor, or the Ki-67 proliferative index. Mucinous carcinomas and right-side carcinomas were less p53 immunoreactive (25% and 52%, respectively) than the usual adenocarcinomas (73%) and distal tumors (72%). These findings suggest that p53 may be a target of chromosome 17 deletions and that this gene may play a role in the malignant transformation of adenomas. BglII and AccII restriction fragment length polymorphism analysis of the p53 gene may be a useful and direct technique to detect allelic loss of this gene in tumors.

Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes.

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.

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.