cAMP-PKA signal transduction specificity in Saccharomyces cerevisiae.

Living cells have developed a set of complex signaling responses, which allow them to withstand different environmental challenges. Signaling pathways enable the cell to monitor external and internal states and to articulate the appropriate physiological responses. Cellular signal transmission requires the dynamic formation of spatiotemporal controlled molecular interactions. One of the most important signaling circuits in Saccharomyces cerevisiae is the one controlled by cAMP-Protein Kinase A (PKA). In budding yeast, extracellular glucose and a plethora of signals related with growth and stress conditions regulate the intracellular cAMP levels that modulate PKA activity which in turn regulates a broad range of cellular processes. The cAMP-PKA signaling output requires a controlled specificity of the PKA responses. In this review we discuss the molecular mechanisms that are involved in the establishment of the specificity in the cAMP-PKA signaling pathway in S.cerevisiae.

Frequency and outcome of total anterior circulation strokes without intracranial large-vessel occlusion.

BACKGROUND AND PURPOSE: The percentage of patients with clinical total anterior circulation infarct (TACI) syndrome treated with reperfusion therapies in the absence of intracranial large-vessel occlusion (ILVO) was determined and their characteristics and outcome are described. METHODS: Data from a population-based, prospective, externally audited registry of all stroke patients treated with intravenous thrombolysis (IVT) and endovascular therapies in Catalonia from January 2011 to December 2013 were used. Patients with a baseline TACI and initial stroke severity measured by the National Institute of Health Stroke Scale (NIHSS) ≥ 8, evaluated less than 4.5 h post-onset, for whom a vascular study prior to treatment was available (n = 1070) were selected. Clinical characteristics, outcome and radiological data for patients treated with IVT alone (n = 605) were compared between those with detected ILVO (n = 474) and non-ILVO patients (n = 131). RESULTS: A total of 1070 patients met study criteria; non-ILVO was found in 131 (12.2%). Analysing the 605 patients treated only with IVT, no significant differences were found between non-ILVO and ILVO patients in age, sex, risk factors, time-to-treatment and type of radiological studies performed. Although non-ILVO patients had lower initial stroke severity (P < 0.001) and a better prognosis (P = 0.001), 51.3% had a poor outcome and 16% were deceased at 90 days. In 66.4% of patients without ILVO, a recent anterior territorial infarct was detected. CONCLUSIONS: Intracranial artery patency was observed in 12.2% of TACI patients evaluated within 4.5 h. Although absence of ILVO was associated with slightly better prognosis, more than half had a poor outcome at 3 months.

DEFB1 gene 5' untranslated region (UTR) polymorphisms are marginally involved in inflammatory bowel disease in south Brazilians.

The possible association of three DEFB1 gene polymorphisms with susceptibility to develop ulcerative colitis (UC) and Crohn's disease (CD) was investigated in Brazilian patients and controls. Although a clear and strong association between functional 5'-UTR DEFB1 SNPs and susceptibility/protection to IBDs cannot be drawn, our results suggest a possible involvement of DEFB1 gene in inflammatory bowel diseases, especially with the colonic localization of Crohn's disease.

Analysis of KIR gene frequencies and HLA class I genotypes in prostate cancer and control group.

Prostate cancer is the second most common cancer in men, with a significant increase in incidence and mortality in men over 50 years of age. Natural killer cells (NK) are part of the innate immune system recognizing class I HLA molecules on target cells through their membrane receptors, called killer cell immunoglobulin-like receptors (KIR). The aim of our study is to evaluate the association between the KIR genes and HLA alleles in patients with prostate cancer and healthy controls. Two hundred patients with prostate cancer and 185 healthy controls were typed for HLA class I and KIR genes by PCR-SSP. When both groups were compared, no significant differences were found for HLA-C group 1 and group 2, HLA-Bw4, HLA-A3 and A11. No difference was seen either in KIR frequency between patients with prostate cancer and controls. In conclusion, our data suggest no potential role for the KIR gene system in prostate cancer.

[Adequacy of the prescription of vitamin D in Primary Care]. / Adecuación de la prescripción de vitamina D en Atención Primaria.

OBJECTIVES: To evaluate the adequacy of vitamin D treatment based on clinic evidence in a Primary Care Center as well as to analyze some characteristics of the prescriptions made. MATERIALS AND METHODS: Descriptive cross-sectional study. Primary Care. Patients above 14 years old with vitamin D prescription. Main variable was the therapeutic adequacy with vitamin D compounds (adequacy was considered when there was a clinical indication for treatment and blood vitamin D levels below 20ng/ml). Other clinical variables were collected. Frequency and association measures were used for statistical analysis. Level of statistical significance was considered <0.05. RESULTS: 430 patients, 346 women (80.5%, 95% CI=77-84). Record of vitamin D values in 216 (50.2%, 95% CI=45-55). Screening/treatment indications in 219 patients (50.9%, 95% CI=46-56), of those in 150 patients vitamin D values were recorded (68.5%, 95% CI=62-75), average (±SD) was 21.22±12ng/ml, deficiency criteria in 86 (57.3%, 95% CI=51-64), insufficiency in 37 (24.7%, 95% CI=19-30) and sufficiency in 27 (18%, 95% CI=13-23). 86 patients (20%, 95% CI=16-24) had treatment indications plus vitamin D deficiency with no differences between genders. CONCLUSIONS: Only 20% of the patients had treatment indications plus vitamin D deficiency. Female predominance. Just over half had indications for screening of serological vitamin D values and/or indications for treatment with vitamin D compounds.

Killer cell immunoglobulin-like receptor gene diversity in a Caucasian population of southern Brazil.

Killer immunoglobulin-like receptors (KIR) regulate the activity of natural killer and T cells through an interaction with specific human leucocyte antigen (HLA) class I molecules on target cells. Diversity in KIR gene content, KIR allelic and haplotype polymorphism has been observed between different ethnic groups. However, most population studies on KIR variability have focused on Europe and Asia, while Americas, Oceania and Africa remain poorly studied. The aim of this study was to analyse the variability of KIR genes in 200 healthy nonrelated individuals from the Southern Brazilian population. KIR genes and HLA-A, -B and -Cw were genotyped using polymerase chain reaction-sequence-specific primers. Southern Brazilian population demonstrated several similarities to states that are closer geographically and distinct differences with Northern Brazil in the frequency of genes KIR2DS1, 2DS2, 2DS3, 2DS5, 3DL1, 3DS1, 2DL1 and 2DL2. The activating gene KIR2DS5 was the least frequent locus found in our group. Interaction of KIR/HLA was more common in the 2DS1-/2DL1+/C2+ association. This study demonstrated the diversity of KIR genes and of KIR/HLA association in a Caucasian group of Southern Brazil, establishing differences and similarities to other different populations.

Last Interglacial Iberian Neandertals as fisher-hunter-gatherers.

Marine food-reliant subsistence systems such as those in the African Middle Stone Age (MSA) were not thought to exist in Europe until the much later Mesolithic. Whether this apparent lag reflects taphonomic biases or behavioral distinctions between archaic and modern humans remains much debated. Figueira Brava cave, in the Arrábida range (Portugal), provides an exceptionally well preserved record of Neandertal coastal resource exploitation on a comparable scale to the MSA and dated to ~86 to 106 thousand years ago. The breadth of the subsistence base-pine nuts, marine invertebrates, fish, marine birds and mammals, tortoises, waterfowl, and hoofed game-exceeds that of regional early Holocene sites. Fisher-hunter-gatherer economies are not the preserve of anatomically modern people; by the Last Interglacial, they were in place across the Old World in the appropriate settings.

[Small vessel cerebrovascular disease]. / Enfermedad cerebrovascular de pequeño vaso.

Small vessel vascular disease is a spectrum of different conditions that includes lacunar infarction, alteration of deep white matter, or microbleeds. Hypertension is the main risk factor, although the atherothrombotic lesion may be present, particularly in large-sized lacunar infarctions along with other vascular risk factors. MRI findings are characteristic and the lesions authentic biomarkers that allow differentiating the value of risk factors and defining their prognostic value.

Adecuación de la prescripción de vitamina D en Atención Primaria / Adequacy of the prescription of vitamin D in Primary Care

Objetivos: Evaluar la adecuación del tratamiento con vitamina D, basada en la evidencia clínica, en un centro de Atención Primaria, así como analizar diversas características de las prescripciones realizadas. Materiales y métodos: Estudio descriptivo transversal. Atención Primaria. Pacientes mayores de 14 años con prescripción de vitamina D. Variable principal fue la adecuación terapéutica con compuestos de vitamina D (se consideró adecuación cuando había indicación clínica de tratamiento y niveles séricos de vitamina D<20 ng/ml). Se recogieron otras variables clínicas. Para el análisis estadístico se utilizaron medidas de frecuencia y asociación. Se consideró un nivel de significación<0,05. Resultados: Cuatrocientos treinta pacientes, 346 mujeres (80,5%, IC-95%=77-84). Registro de valores de vitamina D en 216 (50,2%, IC-95%=45-55). Indicaciones para realizar cribado/tratamiento en 219 pacientes (50,9%, IC-95%=46-56), de los que en 150 (68,5%, IC-95%=62-75) constaban valores de vitamina D, media (±DE) de 21,22±12 ng/ml, criterios de deficiencia en 86 (57,3%, IC-95%=51-64), insuficiencia en 37 (24,7%, IC- 95%=19-30) y suficiencia en 27 (18%, IC-95%=13-23). En 86 pacientes (20%, IC-95%=16-24) había adecuación de tratamiento (indicación de tratamiento sumado a un déficit de vitamina D), sin diferencias entre sexos. Conclusiones: Solo el 20% de los pacientes tratados con vitamina D tenían una buena adecuación terapéutica (indicación para tratamiento junto con un déficit de vitamina D). Había más mujeres con tratamiento. Poco más de la mitad presentaron indicación para cribado analítico de valores serológicos de vitamina D y/o para iniciar tratamiento con fármacos con compuestos de vitamina D. La mitad de los pacientes tenían registro de vitamina D (AU)

Objectives: To evaluate the adequacy of vitamin D treatment based on clinic evidence in a Primary Care Center as well as to analyze some characteristics of the prescriptions made. Materials and methods: Descriptive cross-sectional study. Primary Care. Patients above 14 years old with vitamin D prescription. Main variable was the therapeutic adequacy with vitamin D compounds (adequacy was considered when there was a clinical indication for treatment and blood vitamin D levels below 20ng/ml). Other clinical variables were collected. Frequency and association measures were used for statistical analysis. Level of statistical significance was considered <0.05. Results: 430 patients, 346 women (80.5%, 95% CI=77–84). Record of vitamin D values in 216 (50.2%, 95% CI=45–55). Screening/treatment indications in 219 patients (50.9%, 95% CI=46–56), of those in 150 patients vitamin D values were recorded (68.5%, 95% CI=62–75), average (±SD) was 21.22±12ng/ml, deficiency criteria in 86 (57.3%, 95% CI=51–64), insufficiency in 37 (24.7%, 95% CI=19–30) and sufficiency in 27 (18%, 95% CI=13–23). 86 patients (20%, 95% CI=16–24) had treatment indications plus vitamin D deficiency with no differences between genders. Conclusions: Only 20% of the patients had treatment indications plus vitamin D deficiency. Female predominance. Just over half had indications for screening of serological vitamin D values and/or indications for treatment with vitamin D compounds (AU)

Androgen receptors in human melanoma cell lines IIB-MEL-LES and IIB-MEL-IAN and in human melanoma metastases.

The presence and characteristics of androgen receptors (ARs) have been described by our group in one human melanoma cell line. We have now investigated their presence in two other human melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, as well as in biopsies from human metastatic melanoma. Scatchard analysis revealed a single binding component for both cell lines, the apparent dissociation constant obtained being 15 nM, with a binding capacity of 280 fmol/mg total cell protein, for IIB-MEL-LES cells and 14 nM, with a binding capacity of 206 fmol/mg total cell protein for IIB-MEL-IAN cells. When specificity was assessed, not only androgen and anti-androgen but also non-androgenic compounds were able to compete for [3H]R1881 binding, as seen before. When immunocytochemistry of IIB-MEL-LES and IIB-MEL-IAN cells was performed for ARs, both cell lines were deeply stained in the nucleus, whereas no staining was found for oestrogen or progesterone receptors. Every specimen of melanoma metastases tested for the presence of ARs was deeply stained, and in the majority the intensity of the staining was high. Several hormones and anti-hormones were tested for their ability to affect cell proliferation. In both cell lines, testosterone, dihydrotesterone, oestradiol and progesterone significantly stimulated cell proliferation, and this was reversed by hydroxyflutamide, bicalutamide or tamoxifen.

Monoclonal antibody FC-5.01, directed against CD63 antigen, is internalized into cytoplasmic vesicles in the IIB-BR-G human breast cancer cell line.

Monoclonal antibody (MAb) FC-5.01, raised against the undifferentiated breast cancer cell line IIB-BR-G, has been recently shown to react with CD63. The antigen (Ag) recognized by MAb FC-5.01 is expressed in plasma membranes of IIB-BR-G and other neoplastic cells, as well as in activated platelets and endothelial cells, as detected by indirect immunofluorescence performed at 4 degrees C on live cells. In permeabilized cells, MAb FC-5.01 colocalizes with acridine orange in acidic vesicles (lysosomal/endosomal compartment). Scatchard plot analysis performed on IB-BR-G cells demonstrated a 1.4+/-0.4 x 10(7) M(-1) affinity constant and 2.1 x 10(6) antigenic sites per cell. MAb FC-5.01 is not able to mediate C fixation or ADCC toward CD63+ cells, but the FC-5.01-CD63 complex is efficiently internalized into cytoplasmic vesicles, as shown by an acid wash immunofluorescence assay. Cellular catabolism of the antibody bound by IIB-BR-G cells was studied using [125I]-FC-5.01. At 18 h, >70% of the radioactivity was present in the supernatant as degraded fragments (TCA-soluble). After internalization, rapid Ag re-expression could be demonstrated in IIB-BR-G cells. MAb FC-5.01 diminished migration of CD63+ cells in a Boyden chamber assay. Some of the above-mentioned properties would enable the use of MAb FC-5.01 as a vehicle to target different compounds inside CD63+ cells.

A new epitope on human melanoma-associated antigen CD63/ME491 expressed by both primary and metastatic melanoma.

A monoclonal antibody (MAb) designated FC-5.01 (IgG2a) was generated that binds to several human carcinomas and malignant melanoma. It has revealed no or very low reactivity with most human normal tissues, except for the fact that FC-5.01 binds to some cells from the neuroendocrine system, macrophages, and some renal proximal convolute tubules with an intracellular pattern. Biochemical studies indicate that FC-5.01 recognizes a heterogeneous glycoprotein (broadband between 30-60 kDa) in melanoma tumors. The epitopes reside in the protein core and are presumably conformational, with disulphide bonds implicated in MAb recognition. The current study presents evidence that MAb FC-5.01 reacts with CD63 antigen (Ag), which has been initially described as a melanoma associated Ag, and is a member of the tetraspan family. Reactivity of MAb FC-5.01 with CD63 was demonstrated by Western blot, immunodepletion assay, and FACS analysis of the CD63-negative melanoma cells (KM3) after transfection with the genomic copy of CD63. The epitope recognized by MAb FC-5.01 was shown to be different from the epitope recognized by another anti-CD63 MAb, ME491, by an inhibition radioimmunoassay. Opposite to what has been stated for MAb ME491, no significant differences were found in CD63 expression between primary and metastatic melanoma using MAb FC-5.01.

[Wallenberg's syndrome as the presenting symptom of a cavernous angioma]. / Síndrome de Wallenberg como forma de presentación de un angioma cavernoso.

INTRODUCTION: Cavernous angiomas account for 5-13% of all vascular malformations. In 75% of cases they are situated in the posterior fossa and up to 30% are associated with abnormal venous drainage. The main complication is haemorrhage; the presence of a neurological focus without radiological evidence of bleeding is very rare. CASE REPORT: We report the case of a 54-year-old male with cardiovascular risk factors who presented symptoms that progressively deteriorated over a 72-hour period involving the left lower cranial nerves, sensory impairment and coordination disorder, compatible with Wallenberg's syndrome. Two computerised axial tomography scans of the brain were normal and so a tentative diagnosis of ischemic stroke in progression was proposed. Five days later, magnetic resonance imaging (MR) revealed the presence of a venous angioma and associated abnormal venous drainage. CONCLUSIONS: Cavernous angiomas present a dynamic balance between intracavernous bleeding and thrombosis, with very slow venous blood flow. Upsetting this balance leads to an increase in the intracavernous pressure and involvement of the surrounding tissue, with no radiological expression of bleeding. In these cases MR scanning helps to distinguish between a vascular malformation with reduced blood flow and a clinical picture of ischemic stroke of an arterial origin.

[Cerebral venous thrombosis as the presenting symptom of systemic lupus erythematosus]. / Trombosis venosa cerebral como presentación de lupus eritematoso sistémico.

INTRODUCTION: Cerebral venous thrombosis (CVT) is an infrequent process in systemic lupus erythematosus. We report the case of a female patient whose initial manifestation of lupus was a CVT. CASE REPORT: A 30-year-old female who presented headaches and diminished visual acuity; on exploring the patient bilateral papilloedema was found. Magnetic resonance imaging revealed the presence of a venous thrombosis in the superior and transversal longitudinal sinus. Complementary explorations showed high levels of antinuclear antibodies with leukopenia and proteinuria. Antiphospholipid antibodies were negative. Following treatment with anticoagulants, the patient's condition improved both clinically and radiologically. Months later a biopsy was performed and revealed a grade IV diffuse glomerulonephritis. CONCLUSIONS: In systemic lupus erythematosus, phenomena such as CVT can be the initial form of presentation of the disease. The presence of antiphospholipid antibodies plays a partial role in CVT; other phenomena, such as inflammatory processes, should also be taken into account.

Differential localization to cytoplasm, nucleus or P-bodies of yeast PKA subunits under different growth conditions.

Our aim in this work was to further characterize the complexity and specificity of the three different isoforms (Tpk1, Tpk2 and Tpk3) of the catalytic and regulatory (Bcy1) subunits of PKA in Saccharomyces cerevisiae. We thus analyzed the subcellular localization of the PKA subunits in living cells by using strains carrying GFP (green fluorescent protein) fused to each PKA subunit. During exponential growth on glucose, both Bcy1 and Tpk2 localized in the nucleus, whereas Tpk1 and Tpk3 showed a mixed pattern of nucleo-cytoplasmic localization. During exponential growth on glycerol and during stationary phase, the PKA subunits showed mostly cytoplasmic localization, with the peculiarity that Tpk2 and Tpk3 but not Bcy1, were found associated to P-bodies and EGP bodies. Tpk3 was accumulated into P-bodies during glucose deprivation and hyper osmotic stress. Deletion of Tpk3 altered the kinetics of P-body formation. Analysis of protein expression showed that the relative expression pattern of each Tpk changes from low levels under fermentative metabolism to higher levels during stationary phase. The increase in Tpk levels produced an imbalance with Bcy1 levels. Our data suggest that the signaling specificity through PKA in yeast could be mediated by a particular subcellular localization of each isoform of Tpk.

Enfermedad cerebrovascular de pequeño vaso / Small vessel cerebrovascular disease

La enfermedad vascular de pequeño vaso representa un espectro de diferentes entidades que incluyen el infarto lacunar, la alteración de sustancia blanca y los microsangrados. La hipertensión es el principal factor de riesgo asociado, aunque la lesión aterotrombótica puede estar presente sobre todo en los infartos lacunares de gran tamaño y con otros factores de riesgo vascular. Los hallazgos en RM son característicos y las lesiones auténticos biomarcadores que permiten diferenciar el valor de los factores de riesgo y definir su valor pronóstico

Small vessel vascular disease is a spectrum of different conditions that includes lacunar infarction, alteration of deep white matter, or microbleeds. Hypertension is the main risk factor, although the atherothrombotic lesion may be present, particularly in large-sized lacunar infarctions along with other vascular risk factors. MRI findings are characteristic and the lesions authentic biomarkers that allow differentiating the value of risk factors and defining their prognostic value

[Heterogeneous topography of Foix-Chavany-Marie syndrome]. / Heterogeneidad topográfica del síndrome de Foix-Chavany-Marie.

INTRODUCTION: Bilateral facio-pharyngo-laryngo-glosso-masticatory palsy with automatic-voluntary dissociation is known as Foix-Chavany-Marie (FCM) syndrome. It is usually due to bilateral cortical lesions involving both anterior opercula (biopercular syndrome). We describe three patients with FCM syndrome associated with ischemic lesions in two atypical localizations: a) bilateral subcortical infarcts, and b) unilateral opercular infarct. CASES REPORT: Patient 1, a 66 year old male, was admitted for a sudden onset of anarthria and facial, lingual and masticatory paralysis. Neurological examination revealed automatic-voluntary dissociation of facial motility. MRI showed an acute left subcortical infarct and multiple bilateral subcortical ischemic lesions. Patient 2, a 61 year old male, also suffered a sudden onset of anarthria, with bilateral facial and lingual paralysis and automatic-voluntary dissociation together with sudden onset swallowing alteration. MRI showed a single ischemic lesion involving the left operculum. Patient 3, a 36 year old male, presented sudden onset of dysarthria, dysphagia and bilateral facial palsy with automatic-voluntary dissociation and loss of force in left upper limb. MRI showed an acute right opercular infarct and no contralateral lesions. CONCLUSIONS: FCM syndrome is not only due to bilateral opercular lesions but can also be seen in bilateral subcortical and even unilateral opercular lesions.

Functional properties of FC-2.15, a monoclonal antibody that mediates human complement cytotoxicity against breast cancer cells.

FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15+) cells were investigated. A 51Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15+ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1-50 micrograms/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8 +/- 2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15-. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with 125I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9 x 10(7) M-1 and 2.8 x 10(6) antigenic sites/cell. 125I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15+ cells.