Determination of the appropriate concentration of sodium alginate used for in vitro culture of cat preantral follicles in a serum-free medium containing FSH, EGF and IGF-I.

Culture of domestic cat preantral follicles can be a suitable technology to assist oocyte conservation strategies in the family Felidae. This research was aimed to comparatively analyse cat preantral follicular development of follicles directly seeded on growth surface or encapsulated in 0.5 or 1% of sodium alginate in a serum-free medium containing FSH, EGF and IGF-I. Preantral follicles were isolated from cat ovarian cortical tissue after ovariectomy. Alginate was dissolved at 0.5 or 1% in PBS. Follicles, 4 per well, with 0% (G-0%), 0.5% (G-0.5%) or 1% (G-1%) of sodium alginate were cultured in M199 with FSH (100 ng/mL), EGF (100 ng/mL) and IGF-I (100 ng/mL) for 7 days at 37°C, 5% CO2 and 99% humidity. Culture medium was replaced every 48 h and samples were stored at -20°C until ELISA of steroid hormones. Morphometric evaluation of follicles was performed every 24 h. G-0% follicles showed granulosa cell migration away from the oocyte and disrupted morphology, whereby they reached apparently larger diameters (203.70 ± 5.82 µm; p < .05) than G-0.5% and G-1% follicles (157.89 ± 8.47 µm and 95.23 ± 1.67 µm, respectively) which maintained three-dimensional organization, being larger in G-0.5% than in G-1% (p < .05). G-0.5% follicles attained the multi-layer preantral follicle stage on day 7 of culture, whereas G-1% follicles underwent progressive atresia. On day 6, steroid concentrations were higher (p < .05) in G-0% than in G-1%: 60 ± 19 vs 0.88 ± 0.32 pg/mL oestradiol; 2.6 ± 0.84 vs 0.04 ± 0.02 ng/mL progesterone; 1.3 ± 0.22 vs 0.61 ± 0.04 ng/mL testosterone and 1.6 ± 0.54 vs 0.22 ± 0.07 ng/mL androstenedione respectively. Steroid concentrations in G-0.5% were comprised between those of G-0% and G-1% (p > .05). In conclusion, two-layer cat preantral follicles encapsulated in 0.5% alginate cultured in medium containing FSH, EGF and IGF-I can develop up to the multi-layer preantral stage in 7 days of culture, whereas follicles directly seeded on growth surface or encapsulated in 1% alginate lost their three-dimensional organization, and experienced regression with compromised steroidogenesis, respectively.

Implementation of a virtual laboratory for training on sound insulation testing and uncertainty calculations in acoustic tests.

This paper describes a methodology and case study for the implementation of educational virtual laboratories for practice training on acoustic tests according to international standards. The objectives of this activity are (a) to help the students understand and apply the procedures described in the standards and (b) to familiarize the students with the uncertainty in measurement and its estimation in acoustics. The virtual laboratory will not focus on the handling and set-up of real acoustic equipment but rather on procedures and uncertainty. The case study focuses on the application of the virtual laboratory for facade sound insulation tests according to ISO 140-5:1998 (International Organization for Standardization, Geneva, Switzerland, 1998), and the paper describes the causal and stochastic models and the constraints applied in the virtual environment under consideration. With a simple user interface, the laboratory will provide measurement data that the students will have to process to report the insulation results that must converge with the "virtual true values" in the laboratory. The main advantage of the virtual laboratory is derived from the customization of factors in which the student will be instructed or examined (for instance, background noise correction, the detection of sporadic corrupted observations, and the effect of instrument precision).

Postoperative pain in dogs undergoing either laparoscopic or open ovariectomy.

A prospective, quasi-experimental, clinical trial was performed to assess acute postoperative pain in healthy female dogs following elective ovariectomy by either laparoscopy (n=13) or laparotomy (n=14). Pain was assessed by both a veterinarian at the hospital, and by the owner once the patient was discharged. The Spanish version of the short form of the Glasgow Composite Measuring Pain Scale (CMPS-SF) was used. Pain scores were assessed by the veterinarian preoperatively and at 1, 2, 4, and 6 h after extubation, whilst owner-assessed scores were performed preoperatively and at postoperative days 0, 1, 2, 3, 5 and 7. Data were compared with Mann-Whitney-U test. Veterinarian-assessed CMPS-SF scores were different between both groups at all postoperative times but not at baseline, being below 6/24 in all dogs in the laparoscopy group, but equal to or greater than 6/24 in the laparotomy group at 1 h (n=12), and 4 h (n=4) (P<0.001 and P=0.029, respectively). There were also differences in pain scores between both groups at 2 h (P=0.012) and 6 h (P=0.007), being below 6/24 in all of them. However, there were no differences in owner assessments between groups. In conclusion, ovariectomy performed by laparoscopy induced lower pain scores that were below the pain threshold set by the CMPS-SF during the first 6 h postoperatively. After discharge, and up to one week later, ongoing owner-assessed scores suggest no pain was induced with neither of the techniques. Owners were proactive allowing real-time pain assessment to be reported. The development and validation of instruments for acute pain assessment by owners is warranted, as these tools are currently lacking.

A polymorphism in the regulatory region of APOE associated with risk for Alzheimer's dementia.

The epsilon4 allele of the apolipoprotein E gene (APOE) has been associated with an increased risk of developing Alzheimer's disease (AD; refs 1,2). However, it is apparent that the APOEepsilon4 allele alone is neither necessary nor sufficient to cause the disease. We have recently found three new polymorphisms within the APOE transcriptional regulatory region (M.J.A. et al., manuscript submitted) and now establish an association between one of these polymorphisms (-491A/T) and dementia as observed in Alzheimer's disease, in two independent clinical populations. The results suggest that homozygosity of a common variant (-491A) is associated with increased risk for AD, and that this association is independent of APOEepsilon4 status. In vitro studies suggest that the -491A/T polymorphism may increase risk for AD by altering the level of ApoE protein expression.

Cholesterol dysregulation in peripheral blood mononuclear cells of Alzheimer's disease.

Cholesterol and the immune system are involved in Alzheimer's Disease (AD). To investigate the relations among them, we compared the cholesterol content in peripheral blood mononuclear cells (PBMC) of cognitively healthy controls and patients with mild cognitive impairment (MCI) and AD in two independent samples. Free cholesterol content of PBMC was lower in MCI and AD patients, and was modulated by APOE genotype. A decrease of CD8+ and an increase of CD16+ was also found in AD patients. These results suggest that cholesterol levels in PBMCs may represent an early signature of the disease and support the involvement of immune system in AD.

Assessment of noise exposure during commuting in the Madrid subway.

Because noise-induced hearing impairment is the result not only of occupational noise exposure but also of total daily noise exposure, it is important to take the non-occupational exposure of individuals (during commuting to and from their jobs, at home, and during recreational activities) into account. Mass transit is one of the main contributors to non-occupational noise exposure. We developed a new methodology to estimate a representative commuting noise exposure. The methodology was put into practice for the Madrid subway because of all Spanish subway systems it covers the highest percentage of worker journeys (22.6%). The results of the application highlight that, for Madrid subway passengers, noise exposure level normalized to a nominal 8 hr (L(Ex,8h-cj) ) depends strongly on the type of train, the presence of squealing noise, and the public address audio system, ranging from 68.6 dBA to 72.8 dBA. These values play an important role in a more complete evaluation of a relationship between noise dose and worker health response.

Alzheimer's amyloid precursor protein is expressed on the surface of hematopoietic cells upon activation.

A4-amyloid is the major component of senile plaques and neurofibrillary tangles found in the brain of patients suffering Alzheimer's disease. This 39-42 amino acid peptide is derived from a larger precursor protein (APP). Since APP gene encodes for a putative membrane protein, the study of APP expression at the cell surface may provide useful data for understanding its physiological function. In this report, we present data on APP expression, that was detected by APP specific mAbs in cells of the hematopoietic system. APP was weakly expressed on the cell surface of resting human lymphocytes and monocytes, but it could be induced to the surface of those cells upon stimulation. The cell activators capable of inducing APP membrane expression comprehended mitogenic lectins, calcium ionophores, phosphatase inhibitors, and anti mu-chain or anti-CD3 antibodies in B and T cells, respectively. Interestingly, phorbol esters were able to induce APP membrane expression in monocytic, but not in lymphoid cells. In contrast to lymphocytes and monocytes, granulocytes never expressed cell surface or cytoplasmic APP, even after the activation. The induction of membrane APP in response to lymphocyte activation signals, including antibodies to the antigen receptor of B and T cells, raises the possibility that APP might play the role of a cell surface receptor in the immune system.

Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene.

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides -1017 to +406. Upon a population study, three new polymorphic sites (-491, -427 and -219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites -491 and -219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.

Effect of the Alzheimer amyloid fragment Abeta(25-35) on Akt/PKB kinase and survival of PC12 cells.

The phosphatidylinositol 3 kinase (PI3K)-Akt/PKB pathway protects neurons from apoptosis caused by diverse stress stimuli. However, its protective role against the amyloid beta peptide (Abeta), a major constituent of Alzheimer's disease plaques, has not been studied. We investigated the effect of the Abeta-derived Abeta(25-35) peptide on apoptosis and on the Akt survival pathway in PC12 cells. Cells submitted to micromolar concentrations of Abeta(25-35) exhibited increased production of reactive oxygen species (ROS) and morphological alterations consistent with apoptosis. Akt1 was activated shortly after incubation with Abeta(25-35) and Abeta(1-40) with a kinetics different to that of nerve-derived growth factor. Akt1 activation was blocked by the PI3K inhibitor wortmannin. We tested the hypothesis that Akt1 might modify the vulnerability of neural cells to apoptosis induced by Abeta(25-35). Overexpression of an active version of Akt1 attenuated the apoptotic effect of Abeta(25-35) as determined by flow cytometry. Moreover, PC12 cells overexpressing a membrane-targeted N-myristylated fusion protein of enhanced green fluorescence protein (EGFP) and mouse Akt1 exhibited lower levels of ROS than control EGFP-transfected cells. The present findings demonstrate that Akt1 is activated in response to Abeta(25-35) in a PI3K-dependent manner and that active Akt1 protects PC12 cells against the pro-apoptotic action of this peptide.

Interaction of alpha-2-macroglobulin and HSV-1 during infection of neuronal cells.

We describe the effect of pretreatment with alpha-2-macroglobulin (A2M) on the susceptibility of the human neuroblastoma SKNMC cell line to infection by herpes virus type 1 (HSV-1). ELISA and co-immunoprecipitation experiments confirmed the A2M-HSV-1 interaction in vitro. Indirect immunofluorescence shows that A2M exacerbated the cytopathic effect induced after HSV-1 infection. However, A2M-pretreated SKNMC cells notably produced fewer HSV-1 particles than did the untreated cells, suggesting that A2M could induce a restrictive infection. Furthermore, high levels of HSV-1 and A2M induced the production of nitric oxide (NO) in SKNMC. Preliminary results suggest that A2M might induce apoptosis in HSV-1-infected cells. These findings affirm the conclusion that A2M may interact directly with HSV-1 and modulate the course of the infection in SKNMC human neuroblastoma cells.

Risk for Alzheimer's disease correlates with transcriptional activity of the APOE gene.

While the straightepsilon4 allele of apolipoprotein E ( APOE, gene; ApoE, protein) is widely accepted as a major genetic risk factor for the late onset form of Alzheimer's disease (AD), recent evidence points to variations in ApoE levels as another important factor. We have previously reported that a common variant in the regulatory region of APOE (-491A) is associated with risk for late onset AD. In this report we analyze the association of another APOE promoter polymorphism (-427T/C) with AD in two case-control clinical samples and demonstrate a correlation between APOE promoter transcriptional activity and risk for AD. The association studies show that the allelic variant (-427C) and the haplotype [-491A-427C] of the APOE promoter are associated with increased risk for AD. Study of the transcriptional activity of the common haplotypes defined by combination of the -491 and -427 alleles indicated that the risk for late onset AD positively correlates with transcriptional activity of the APOE gene, suggesting that increases in the local expression of ApoE could be responsible for the association of APOE promoter polymorphism with AD.

Lipoma intraarticular con presentación clínica de meniscopatía / Intra-articular lipoma with clinical presentation of meniscal disease

Presentamos el caso de un lipoma de localización articular, concretamente en rodilla. La literatura define estos tumores como extremadamente infrecuentes y es el único publicado cuya presentación semeja la clínica y la exploración típica de una meniscopatía. Analizamos la bibliografía existente, que diferencia dos tipos de tumoraciones benignas de tejido adiposo en la rodilla: el lipoma arborescente, más frecuente, y el lipoma intraarticular, que es nuestro caso, mucho más raro. Comentamos los datos acerca de su etiología, forma de presentación, diagnóstico y tratamiento. Concluimos que los tumores benignos de rodilla en general y los lipomas intraarticulares en particular deben ser tenidos en cuenta en el diagnóstico diferencial de los cuadros de dolor mecánico en rodilla(AU)

We report the case of a Lipoma located in the joint, specifically in the knee. The literature defines these tumors as extremely rare and this is the first one to be published that simulates the clinical presentation and typical examination of a knee meniscal disease. We analyzed the existing literature, which distinguishes two types of benign tumors of adipose tissue in the knee (the Arborescens Lipoma, which is the most common, and the intra-articular Lipoma, which is our case, and is much rarer). We have discussed the data on its etiology, clinical presentation, diagnosis and treatment. We conclude that benign tumors of the knee, in general, and particularly intra-articular Lipoma should be considered in the differential diagnosis of the cases of mechanical knee pain(AU)