A link between FXYD3 (Mat-8)-mediated Na,K-ATPase regulation and differentiation of Caco-2 intestinal epithelial cells.

FXYD3 (Mat-8) proteins are regulators of Na,K-ATPase. In normal tissue, FXYD3 is mainly expressed in stomach and colon, but it is also overexpressed in cancer cells, suggesting a role in tumorogenesis. We show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and a decrease in transepithelial resistance. Inhibition of cell differentiation in FXYD3-deficient cells is accompanied by an increase in the apparent Na+ and K+ affinities of Na,K-ATPase, reflecting the absence of Na,K-pump regulation by FXYD3. In addition, we observe a decrease in the maximal Na,K-ATPase activity due to a decrease in its turnover number, which correlates with a change in Na,K-ATPase isozyme expression that is characteristic of cancer cells. Overall, our results suggest an important role of FXYD3 in cell differentiation of Caco-2 cells. One possibility is that FXYD3 silencing prevents proper regulation of Na,K-ATPase, which leads to perturbation of cellular Na+ and K+ homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell differentiation.

Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.

Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

Assessment of soil properties by organic matter and EM-microorganism incorporation

Properties of a claim loam soil, collected in Aranjuez (Madrid) and enriched with organic matter and microorganisms, were evaluated under controlled temperature and moisture conditions, over a period of three months. The following treatments were carried out: soil (control); soil + 50 t ha-1 of animal manure (E50); soil + 50 t ha-1 of animal manure + 30 L ha-1 of effective microorganisms (E50EM); soil + 30 t ha-1 of the combination of various green crop residues and weeds (RC30) and soil + 30 t ha-1 of the combination of various green crop residues and weeds + 30 L ha-1 of effective microorganisms (RC30EM). Soil samples were taken before and after incubation and their physical, chemical, and microbiological parameters analyzed. Significant increase was observed in the production of exopolysaccharides and basic phosphatase and esterase enzyme activities in the treatments E50EM and RC30EM, in correlation with the humification of organic matter, water retention at field capacity, and the cationic exchange capacity (CEC) of the same treatments. The conclusion was drawn that the incorporation of a mixture of effective microorganisms (EM) intensified the biological soil activity and improved physical and chemical soil properties, contributing to a quick humification of fresh organic matter. These findings were illustrated by the microbiological activities of exopolysaccharides and by alkaline phosphatase and esterase enzymes, which can be used as early and integrated soil health indicators.

Population-based genome-wide association studies reveal six loci influencing plasma levels of liver enzymes.

Plasma liver-enzyme tests are widely used in the clinic for the diagnosis of liver diseases and for monitoring the response to drug treatment. There is considerable evidence that human genetic variation influences plasma levels of liver enzymes. However, such genetic variation has not been systematically assessed. In the present study, we performed a genome-wide association study of plasma liver-enzyme levels in three populations (total n = 7715) with replication in three additional cohorts (total n = 4704). We identified two loci influencing plasma levels of alanine-aminotransferase (ALT) (CPN1-ERLIN1-CHUK on chromosome 10 and PNPLA3-SAMM50 on chromosome 22), one locus influencing gamma-glutamyl transferase (GGT) levels (HNF1A on chromosome 12), and three loci for alkaline phosphatase (ALP) levels (ALPL on chromosome 1, GPLD1 on chromosome 6, and JMJD1C-REEP3 on chromosome 10). In addition, we confirmed the associations between the GGT1 locus and GGT levels and between the ABO locus and ALP levels. None of the ALP-associated SNPs were associated with other liver tests, suggesting intestine and/or bone specificity. The mechanisms underlying the associations may involve cis- or trans-transcriptional effects (some of the identified variants were associated with mRNA transcription in human liver or lymphoblastoid cells), dysfunction of the encoded proteins (caused by missense variations at the functional domains), or other unknown pathways. These findings may help in the interpretation of liver-enzyme tests and provide candidate genes for liver diseases of viral, metabolic, autoimmune, or toxic origin. The specific associations with ALP levels may point to genes for bone or intestinal diseases.

Microbial and enzymatic activity in soil after organic composting

Microbial activity and biochemical properties are important indicators of the impact of organic composting on soil. The objective of this study was to evaluate some indicators of soil microbial and biochemical processes after application of compost (household waste). A Typic Acrustox, sampled at a depth of 10 cm under Cerrado biome vegetation, was evaluated in three treatments: control (soil without organic compost amendment) and soil with two doses of domestic organic compost (10 and 20 g kg-1 soil). The following properties were evaluated: released C (C-CO2): microbial respiration 15 days after incubation; microbial biomass C (MBC); total glucose (TG); metabolic quotient (qCO2); and enzyme activity of β-glucosidase and acid and alkaline phosphatase. The application of household compost, at doses of 10 and 20 g kg-1 Typic Acrustox, resulted in significant gains in microbial activity, organic C and C stock, as evidenced by increased MBC and TG levels. On the other hand, qCO2 decreases indicated greater microbial diversity and more efficient energy use. The addition of compost, particularly the 20 g kg-1 dose, strongly influenced the enzyme β-glucosidase and phosphatase (acid and alkaline). The β-glucosidase activity was significantly increased and acid phosphatase activity increased more than the alkaline. The ratio of β-glucosidase to MBC was greater in the control than in the composted treatments which suggests that there were more enzymes in the control than in the substrate or that the addition of compost induced a great MBC increase.

Cardiorespiratory arrest and vitamin D deficiency rickets: A case report

Vitamin D deficiency rickets became a rare disease in industrialized countries due to vitamin D supplementation in infants and nutritional guidelines. Symptoms of hypocalcemia due to vitamin D deficiency rickets may be life threatening. We report a case of a 16 months old infant who initially presented with stridor that was misdiagnosed as viral laryngitis. He presented, two weeks later, a cardiorespiratory arrest related to a laryngospasm secondary to severe hypocalcemia (ionized calcium level: 0.42 mmol/l,total calcium level: 1.15 mmol/). He was successfully resuscitated and vitamin D deficiency rickets was diagnosed. The medical history revealed that the infant was exclusively breast fed without vitamin D supplementation till the age of 10 months and also deprived from other milk products intentionally by the parents due to cultural habits. The laboratory investigations showed an elevated alkaline phosphatase level at 577 U/l, a normal phosphatemia level at 2 mmol/l, a decreased 25 (OH) cholecalciferol at 5.7 mcg/l,a normal calciuria level at 0.35 mol/mol of creatinine and an increased parathyroid hormone level at 325 ng/l. Cardiocirculatory arrest secondary to vitamin D deficiency rickets is very rare. The aim of this presentation is to highlight the symptoms of vitamin D deficiency rickets and to raise pediatricians' awareness to the necessity of including the diagnosis of hypocalcemia in case of stridor especially if the nutritional history or ethnic origin of the infant predispose to vitamin D deficiency. Vitamin D supplementation is important for some ethnic minority population, whom are faced with the risk of developing this disease

High prevalence of severe vitamin D deficiency in combined antiretroviral therapy-naive and successfully treated Swiss HIV patients.

OBJECTIVES: To evaluate the prevalence of 25-hydroxyvitamin D [25(OH)D] deficiency in HIV-positive patients, a population at risk for osteoporosis. DESIGN: Retrospective assessment of vitamin D levels by season and initiation of combined antiretroviral therapy (cART). METHODS: 25(OH)D was measured in 211 HIV-positive patients: samples were taken before initiation of cART from February to April or from August to October as well as 12 (same season) and 18 months (alternate season) after starting cART. 1,25-Dihydroxyvitamin D [1,25(OH)2D] was measured in a subset of 74 patients. Multivariable analyses included season, sex, age, ethnicity, BMI, intravenous drug use (IDU), renal function, time since HIV diagnosis, previous AIDS, CD4 cell count and cART, in particular nonnucleoside reverse transcriptase inhibitor (NNRTI) and tenofovir (TDF) use. RESULTS: At baseline, median 25(OH)D levels were 37 (interquartile range 20-49) nmol/l in spring and 57 (39-74) nmol/l in the fall; 25(OH)D deficiency less than 30 nmol/l was more prevalent in spring (42%) than in fall (14%), but remained unchanged regardless of cART exposure. In multivariable analysis, 25(OH)D levels were higher in white patients and those with a longer time since HIV diagnosis and lower in springtime measurements and in those with active IDU and NNRTI use. 1-Hydroxylation rates were significantly higher in patients with low 25(OH)D. Hepatitis C seropositivity, previous AIDS and higher CD4 cell counts correlated with lower 1,25(OH)2D levels, whereas BMI and TDF use were associated with higher levels. In TDF-treated patients, higher 1,25(OH)2D correlated with increases in serum alkaline phosphatase. CONCLUSION: Based on the high rate of vitamin D deficiency in HIV-positive patients, systematic screening with consideration of seasonality is warranted. The impact of NNRTIs on 25(OH)D and TDF on 1,25(OH)2D needs further attention.

Microbiological and biochemical properties of an agricultural Mexican soil amended with sewage sludge

The application of sewage sludge is a concern because it may affect the quality of organic matter and microbiological and biochemical soil properties. The effects of surface application of sewage sludge to an agricultural soil (at 18 and 36 t ha-1 dry basis) were assessed in one maize (Zea mays L.) growing season. The study evaluated microbial biomass, basal respiration and selected enzymatic activities (catalase, urease, acid and alkaline phosphatase, and β-glucosidase) 230 days after sewage sludge application and infrared spectroscopy was used to assess the quality of dissolved organic matter and humic acids. Sewage sludge applications increased the band intensity assigned to polysaccharides, carboxylic acids, amides and lignin groups in the soil. The organic matter from the sewage sludge had a significant influence on the soil microbial biomass; nevertheless, at the end of the experiment the equilibrium of the soil microbial biomass (defined as microbial metabolic quotient, qCO2) was recovered. Soil urease, acid and alkaline phosphatase activity were strongly influenced by sewage sludge applications.

Bone metabolism and risk of secondary hyperparathyroidism 12 months after gastric banding in obese pre-menopausal women.

OBJECTIVE: To evaluate, during the first postoperative year in obese pre-menopausal women, the effects of laparoscopic gastric banding on calcium and vitamin D metabolism, the potential modifications of bone mineral content and bone mineral density, and the risk of development of secondary hyperparathyroidism. SUBJECTS: Thirty-one obese pre-menopausal women aged between 25 and 52 y with a mean body mass index (BMI) of 43.6 kg/m(2), scheduled for gastric banding were included. Patients with renal, hepatic, metabolic and bone disease were excluded. METHODS: Body composition and bone mineral density (BMD) were measured at baseline, 6 and 12 months after gastric banding using dual-energy X-ray absorptiometry. Serum calcium, phosphate, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, bilirubin, urea, creatinine, uric acid, proteins, parathormone, vitamin D(3), IGF-1, IGF-BP3 and telopeptide, as well as urinary telopeptide, were measured at baseline and 1, 3, 6, 9 and 12 months after surgery. RESULTS: After 1 y vitamin D3 remained stable and PTH decreased by 12%, but the difference was not significant. Serum telopeptide C increased significantly by 100% (P<0.001). There was an initial drop of the IGF-BP3 during the first 6 months (P<0.05), but the reduction was no longer significant after 1 y. The BMD of cortical bone (femoral neck) decreased significantly and showed a trend of a positive correlation with the increase of telopeptides (P<0.06). The BMD of trabecular bone, at the lumbar spine, increased proportionally to the reduction of hip circumference and of body fat. CONCLUSION: There is no evidence of secondary hyperparathyroidism 1 y after gastric banding. Nevertheless biochemical bone markers show a negative remodelling balance, characterized by an increase of bone resorption. The serum telopeptide seems to be a reliable parameter, not affected by weight loss, to follow up bone turnover after gastroplasty.

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease.

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.

Sandwich enzyme immunoassay using three monoclonal antibodies against different epitopes of carcinoembryonic antigen (CEA).

Purified monoclonal antibodies (Mab) produced by 3 hybridomas and reacting with 3 different epitopes of carcinoembryonic antigen (CEA) were used in a solid phase enzyme immunoassay. Two Mabs were physically adsorbed to polystyrene balls and the third Mab was coupled to alkaline phosphatase using the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio)-propionate. During a first incubation, CEA from heat-extracted serum samples was immunoadsorbed to the antibody coated balls. After washing of the balls, bound CEA was detected by a second incubation with the enzyme coupled Mab. The sensitivity of the assay was 0.6 ng per ml of serum. A total of 196 serum samples from patients with various types of carcinoma, with liver cirrhosis, or from healthy blood donors with or without smoking habits, were tested. The results obtained with the monoclonal enzyme immunoassay (M-EIA) were compared with those obtained with perchloric acid extracts of the same serum samples tested by an inhibition radioimmunoassay using conventional goat anti-CEA antiserum. There was an excellent correlation between the two assays. In particular, the new M-EIA gave good results for the detection of tumor recurrences in the follow-up of colon carcinoma patients. However, despite the use of exclusively monoclonal antibodies the new assay detected a similar percentage of slightly elevated CEA values as the conventional assay in patients with non-malignant disease, suggesting that the CEA associated with non-malignant diseases is immunologically identical to the CEA released by colon carcinoma.

L'ostéolyse expansive familiale : manifestations otologiques et dentaires d'origine génétique

RESUME Nous rapportons l'étude d'une famille de 49 membres sur 5 générations. Parmi 35 membres étudiés, 18 sont atteints d'Osteolyse Expansive Familiale (OEF). L'OEF est une dysplasie osseuse génétique rare, autosomique dominante, dont les altérations locales et générales du squelette ont une distribution périphérique prédominante qui devient manifeste à partir de la deuxième décennie de vie. Une résorption ostéoclastique progressive, accompagnée d'une faible activité ostéoblastique, est à l'origine d'une expansion médullaire osseuse. Cette dernière est caractérisée par une raréfaction de la moelle osseuse qui est remplacée par du tissu fibreux et de la graisse. L'amincissement de la moelle osseuse aboutit à des déformations invalidantes, sévères et douloureuses du squelette, avec tendance aux fractures spontanées. La première manifestation clinique de la maladie est une surdité de transmission très précoce résultant d'une lyse de la chaîne ossiculaire. Radiologiquement, il existe toujours une pneumatisation marquée de la mastoïde et du rocher. Les dents montrent des signes importants de résorption osseuse au niveau de la région apicale et/ou du collet, dont l'aspect est caractéristique et unique. La phosphatase alcaline sérique, l'hydroxyproline et la deoxypiridoline urinaire sont élevées à des taux variables. Le taux de calcium et d'hormone parathyroïdienne est normal. Le traitement par les diphosphonates, la calcitonine et la vitamine D est inefficace. Histologiquement, l'OEF présente des similitudes avec la maladie de Paget, mais l'âge de début, la distribution des lésions osseuses, les altérations dentaires et de l'oreille moyenne, ainsi que la progression clinique sont différents. Il en va de même pour la dysplasie fibreuse, l'ostéite fibro-kystique et l'ostéogénèse imparfaite. Le gêne responsable de la maladie se localise dans la région du chromosome 18q21-22. Récemment, des mutations du TNFRSF 11A, gêne qui codifie le RANK, ont été identifiées comme étant la cause de l'OEF. La duplication de la 18ème paire de base au niveau de l'exon 1 suggère qu'il correspond au site de l'anomalie. La technique chirurgicale et les résultats audiométriques à court et long terme de 13 interventions chez 8 patients sont présentés. ABSTRACT Objectives: Familial Expansive Osteolysis (EEO) is a rare autosomal dominant bone dys¬plasia. The disease can show general and focal skeletal alterations, the latter having a pre¬dominantly peripheral distribution. Onset occurs after the second decade of life. Patients and methods: We present the study, of 30 years, of a family consisting of 49 members covering five generations. Results: Among the 35 members studied, 18 have familial expansive osteolysis (FEO). The first clinical sign of the condition is transmission deafness at an early age. The features of the teeth has a unique and characteristic appearance. Thinning of the corti¬cal bone leads to severe, painful, disabling deformities. Serum alkaline phosphatase, and urinary hydroxyproline and deoxipyridinoline are elevated. Calcium and parathyroid hor¬mone are normal. Treatment with diphosphonates, calcitonin and vitamin D has been unsuccessful. We present the surgical technology and the results to short and long term of 13 interventions on 8 patients. Conclusion: Progressive osteoclastic reabsorption accompanied by weak osteoblastic activ¬ity results in medullary expansion characterized by rarefaction of the bone marrow, which is replaced by fibrous tissue and fat. FE0 is histologically similar to Paget disease, but the age of onset, the distribution of the bone lesions, the dental and middle ear alterations, and the clin¬ical progression are different. These features also differentiate FE0 from fibrous dysplasia, fibrocystic osteitis and imperfect osteogenesis. The gene responsible for EEO is located in the 18q21-22 chromosome region. Mutations in TNFRSF11A, the gene encoding receptor activa¬tor of nuclear factor-kappa-B (RANK), has been recently identified as the cause of FEO. A duplication of 18 base pairs in exon 1 of the TNFRSF11A gene suggests that this corresponds to the site of the anomaly and can be considered a "hot spot" for mutations.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Effect of supplementation with vitamin D3 and calcium on quantitative ultrasound of bone in elderly institutionalized women: a longitudinal study.

Supplementation of elderly institutionalized women with vitamin D and calcium decreased hip fractures and increased hip bone mineral density. Quantitative ultrasound (QUS) measurements can be performed in nursing homes, and easily repeated for follow-up. However, the effect of the correction of vitamin D deficiency on QUS parameters is not known. Therefore, 248 institutionalized women aged 62-98 years were included in a 2-year open controlled study. They were randomized into a treated group (n = 124), receiving 440 IU of vitamin D3 combined with 500 mg calcium (1250 mg calcium carbonate, Novartis) twice daily, and a control group (n = 124). One hundred and three women (42%), aged 84.5 +/- 7.5 years, completed the study: 50 in the treated group, 53 in the controls. QUS of the calcaneus, which measures BUA (broadband ultrasound attenuation) and SOS (speed of sound), and biochemical analysis were performed before and after 1 and 2 years of treatment. Only the results of the women with a complete follow-up were taken into account. Both groups had low initial mean serum 25-hydroxyvitamin D levels (11.9 +/- 1.2 and 11.7 +/- 1.2 micrograms/l; normal range 6.4-40.2 micrograms/l) and normal mean serum parathyroid hormone (PTH) levels (43.1 +/- 3.2 and 44.6 +/- 3.5 ng/l; normal range 10-70 ng/l, normal mean 31.8 +/- 2.3 ng/l). The treatment led to a correction of the metabolic disturbances, with an increase in 25-hydroxyvitamin D by 123% (p < 0.01) and a decrease in PTH by 18% (p < 0.05) and of alkaline phosphatase by 15% (p < 0.01). In the controls there was a worsening of the hypovitaminosis D, with a decrease of 25-hydroxyvitamin D by 51% (p < 0.01) and an increase in PTH by 51% (p < 0.01), while the serum calcium level decreased by only 2% (p < 0.01). After 2 years of treatment BUA increased significantly by 1.6% in the treated group (p < 0.05), and decreased by 2.3% in the controls (p < 0.01). Therefore, the difference in BUA between the treated subjects and the controls (3.9%) was significant after 2 years (p < 0.01). However, SOS decreased by the same amount in both groups (approximately 0.5%). In conclusion, BUA, but not SOS, reflected the positive effect on bone of supplementation with calcium and vitamin D3 in a population of elderly institutionalized women.

Characterization of MAP1B heavy chain interaction with actin.

Microtubule-associated protein 1B is an essential protein during brain development and neurite outgrowth and was studied by several assays to further characterize actin as a major interacting partner. Tubulin and actin co-immunoprecipitated with MAP1B at similar ratios throughout development. Their identity was identified by mass spectrometry and was confirmed by Western blots. In contrast to previous reports, the MAP1B-actin interaction was not dependent on the MAP1B phosphorylation state, since actin was precipitated from brain tissue throughout development at similar ratios and equal amounts were precipitated before and after dephosphorylation with alkaline phosphatase. MAP1B heavy chain was able to bind actin directly and therefore the N-terminal part of MAP1B heavy chain must also contain an actin-binding site. The binding force of this interaction was measured by atomic force microscopy and values were in the same range as those of MAP1B binding to tubulin or that measured in MAP1B self-aggregation. Aggregation was confirmed by negative staining and electron microscopy. Experiments including COS-7 cells, PC12 cells, cytochalasin D and immunocytochemistry with subsequent confocal laser microscopy, suggested that MAP1B may bind to actin but has no obvious microfilament stabilizing effect. We conclude, that the MAP1B heavy chain has a microtubule-stabilization effect, and contains an actin-binding site that may play a role in the crosslinking of actin and microtubules, a function that may be important in neurite elongation.