Raloxifene modulates regulators of osteoclastogenesis and angiogenesis in an oestrogen deficiency periapical lesion model

Aim To analyse the local regulatory mechanisms of osteoclastogenesis and angiogenesis during the progression of periapical lesions in female rats with oestrogen deficiency and treatment with raloxifene (RLX). Methodology Female Wistar rats were distributed into groups: SHAM-veh, subjected to sham surgery and treated with a vehicle; OVX-veh, subjected to ovary removal and treated with a vehicle; and OVX-RLX, subjected to ovary removal and treated with RLX. Vehicle or RLX was administered orally for 90 days. During treatment, the dental pulp of mandibular first molars was exposed to the oral environment for induction of periapical lesions, which were analysed after 7 and 30 days. After the experimental periods, blood samples were collected for measurement of oestradiol, calcium, phosphorus and alkaline phosphatase. The rats were euthanized and the mandibles removed and processed for immunohistochemical detection of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), hypoxia-inducible factor-1 alpha (HIF-1α) and bone-specific alkaline phosphatase (BALP). Data were compared using Kruskal–Wallis followed by Dunn test (nonparametric values) and anova followed by the Tukey's test (parametric values). Results The plasma concentration of oestradiol showed hypo-oestrogenism in the rats subjected to ovary removal. On day 7, alkaline phosphatase activity, calcium and phosphorus were higher in the OVX-RLX group than in the OVX-veh group (P < 0.001), but immunolabelling for RANKL and HIF-1α was lower in OVX-RLX group (P < 0.001). On day 30, the OVX-veh group had higher immunolabelling for RANKL than the OVX-RLX group (P < 0.05). There were no significant differences in the immunoreactivity of OPG and BALP between any groups at either time-point (P > 0.05). Conclusion RLX therapy reversed the increased levels of the local regulators of both osteoclastogenesis and angiogenesis induced by oestrogen deficiency.

Field and laboratory studies to assess the effects of Vertimec (R) 18EC on Daphnia similis

The present study aimed to evaluate the interactions of the pesticide Vertimec (R) 18EC in aquatic ecosystems. In this respect, soil plots were contaminated with Vertimec (R) 18EC at the concentration indicated for strawberry crops (0.125 L of solution m(-2)). After the contamination, torrential rainfall was simulated and the surface runoff was collected and transferred to mesocosm tanks in five treatments, run in triplicate: (1) control-C; (2) runoff from an uncontaminated plot-UR; (3) runoff from the plot contaminated with Vertimec (R) 18EC-CR; (4) direct application of Vertimec (R) 18EC in the water-V and (5) water samples gathered randomly to verify whether there was contamination between the mesocosms-RS. Water samples from these tanks were also submitted to ecotoxicological tests with Daphnia similis and analyses to evaluate the limnological characteristics, in five collection periods over 10 days (240 h). Physical and chemical differences were observed in the water samples, mainly related to increased turbidity, suspended solids and nutrients (nitrogen and phosphate forms). Acute toxicity was observed for the direct application treatment for the entire experimental period, and in some periods for the CR treatment (from 48 h to 168 h). The results obtained suggest that the pesticide did not fully degrade during the study period (10 days) in the direct application treatment, demonstrating that the presence of other substances in the commercial formulation contribute to the maintenance of toxicity. This represents a potential risk for aquatic ecosystems in areas adjacent to where the chemical is applied. (C) 2011 Elsevier Inc. All rights reserved.

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.

Prevalence of vitamin B12 depletion and deficiency in Liechtenstein

OBJECTIVE: Data about vitamin B12 (B12) deficiency in the general population are scarce. The present study was performed to determine the prevalence of B12 deficiency in the general population of the Principality of Liechtenstein, as well as to identify sub-populations potentially at high risk. DESIGN: Retrospective study. SETTING: Ambulatory setting, population of the Principality of Liechtenstein. SUBJECTS: Seven thousand four hundred and twenty-four patients seeking medical attention whose serum samples were referred for routine work-up in an ambulatory setting were consecutively enrolled. Serum total B12 was determined in all patients in this cohort. In addition, for a subgroup of 1328 patients, serum holotranscobalamin was also measured. Prevalence of B12 deficiency was calculated. Further, multivariate logistical regression models were applied to identify covariates independently associated with B12 deficiency and depletion. RESULTS: Nearly 8 % of the general population was suffering from either B12 depletion or deficiency. The ratio between B12 depletion and deficiency was 2:1 for all age ranges. Pathological changes were detected predominantly in older people. Female gender was a significant predictor of B12 depletion. In the cohort, nearly 40 % exhibited either depletion or deficiency of B12. CONCLUSIONS: B12 depletion and deficiency are common in Liechtenstein, a Central European country. The measurement of biochemical markers represents a cost-efficient and valid assessment of the B12 state. When a deficiency of B12 is diagnosed at an early stage, many cases can be treated or prevented, with beneficial effects on individual outcomes and subsequent potential reductions in health-care costs.

Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships

Copper (Cu) and its alloys are used extensively in domestic and industrial applications. Cu is also an essential element in mammalian nutrition. Since both copper deficiency and copper excess produce adverse health effects, the dose-response curve is U-shaped, although the precise form has not yet been well characterized. Many animal and human studies were conducted on copper to provide a rich database from which data suitable for modeling the dose-response relationship for copper may be extracted. Possible dose-response modeling strategies are considered in this review, including those based on the benchmark dose and categorical regression. The usefulness of biologically based dose-response modeling techniques in understanding copper toxicity was difficult to assess at this time since the mechanisms underlying copper-induced toxicity have yet to be fully elucidated. A dose-response modeling strategy for copper toxicity was proposed associated with both deficiency and excess. This modeling strategy was applied to multiple studies of copper-induced toxicity, standardized with respect to severity of adverse health outcomes and selected on the basis of criteria reflecting the quality and relevance of individual studies. The use of a comprehensive database on copper-induced toxicity is essential for dose-response modeling since there is insufficient information in any single study to adequately characterize copper dose-response relationships. The dose-response modeling strategy envisioned here is designed to determine whether the existing toxicity data for copper excess or deficiency may be effectively utilized in defining the limits of the homeostatic range in humans and other species. By considering alternative techniques for determining a point of departure and low-dose extrapolation (including categorical regression, the benchmark dose, and identification of observed no-effect levels) this strategy will identify which techniques are most suitable for this purpose. This analysis also serves to identify areas in which additional data are needed to better define the characteristics of dose-response relationships for copper-induced toxicity in relation to excess or deficiency.

Oral SCH 39304 as primary, salvage, and maintenance therapy for cryptococcal meningitis in AIDS

To determine the efficacy and toxicity of SCH 39304 in the treatment and suppression of cryptococcal meningitis, we conducted a prospective, noncomparative study in three groups of patients: patients with acute cryptococcal meningitis, patients with acute cryptococcal meningitis in whom other therapies have failed (salvage), and patients who required maintenance therapy. As primary therapy, the patients received up to 14 days or 1 g of amphotericin B followed by SCH 39304 200 mg once daily for 12 weeks. As maintenance therapy, the patients received SCH 39304 600 mg once weekly for 12 months. Of five salvage patients, none completed the study. Two patients died, two patients clinically deteriorated, and one patient was noncompliant. Two of three patients with acute cryptococcal meningitis completed the 12-week primary therapy, and one patient was discontinued from therapy because of a skin rash (95% confidence interval, 14-100%). All four patients who were receiving weekly maintenance therapy followed up to 27 weeks were clinically stable with no change in their serum cryptococcal antigen titer from baseline when the study was prematurely terminated. Elevation of liver function test results developed in three patients and skin rash developed in one patient. The unique pharmacologic and pharmacokinetic properties of SCH 39304 (low incidence of toxicity, long serum half-life, and good penetration into the cerebrospinal fluid) lend promise to pursue other triazole antifungals at higher doses as primary therapy and less frequent dosing for maintenance therapy.

Hypermethylation of the DPYD promoter region is not a major predictor of severe toxicity in 5-fluorouracil based chemotherapy

BACKGROUND: The activity of dihydropyrimidine dehydrogenase (DPD), the key enzyme of pyrimidine catabolism, is thought to be an important determinant for the occurrence of severe toxic reactions to 5-fluorouracil (5-FU), which is one of the most commonly prescribed chemotherapeutic agents for the treatment of solid cancers. Genetic variation in the DPD gene (DPYD) has been proposed as a main factor for variation in DPD activity in the population. However, only a small proportion of severe toxicities in 5-FU based chemotherapy can be explained with such rare deleterious DPYD mutations resulting in severe enzyme deficiencies. Recently, hypermethylation of the DPYD promoter region has been proposed as an alternative mechanism for DPD deficiency and thus as a major cause of severe 5-FU toxicity. METHODS: Here, the prognostic significance of this epigenetic marker with respect to severe 5-FU toxicity was assessed in 27 cancer patients receiving 5-FU based chemotherapy, including 17 patients experiencing severe toxic side effects following drug administration, none of which were carriers of a known deleterious DPYD mutation, and ten control patients. The methylation status of the DPYD promoter region in peripheral blood mononuclear cells was evaluated by analysing for each patient between 19 and 30 different clones of a PCR-amplified 209 base pair fragment of the bisulfite-modified DPYD promoter region. The fragments were sequenced to detect bisulfite-induced, methylation-dependent sequence differences. RESULTS: No evidence of DPYD promoter methylation was observed in any of the investigated patient samples, whereas in a control experiment, as little as 10% methylated genomic DNA could be detected. CONCLUSION: Our results indicate that DYPD promoter hypermethylation is not of major importance as a prognostic factor for severe toxicity in 5-FU based chemotherapy.

Pica and refractory iron deficiency anaemia: a case report

ABSTRACT: INTRODUCTION: Iron deficiency is the most common cause of anaemia worldwide. Pica, the ingestion of substances that are inappropriate for consumption, is associated with iron deficiency and may be under-diagnosed. CASE PRESENTATION: A 34-year-old woman presented with iron deficiency anaemia refractory to treatment for more than a decade. The clinical presentation, endoscopic findings and laboratory investigations were consistent with pica. Subsequent geophysical analysis confirmed that the ingested material was kaolin, a negatively charged silicate. CONCLUSION: Prolonged unexplained iron deficiency anaemia should prompt clinicians to remember and inquire about pica. In our patient, this would have averted numerous unnecessary investigations and prevented a decade-long suffering.

Mutations in SDHD lead to autosomal recessive encephalomyopathy and isolated mitochondrial complex II deficiency

BACKGROUND Defects of the mitochondrial respiratory chain complex II (succinate dehydrogenase (SDH) complex) are extremely rare. Of the four nuclear encoded proteins composing complex II, only mutations in the 70 kDa flavoprotein (SDHA) and the recently identified complex II assembly factor (SDHAF1) have been found to be causative for mitochondrial respiratory chain diseases. Mutations in the other three subunits (SDHB, SDHC, SDHD) and the second assembly factor (SDHAF2) have so far only been associated with hereditary paragangliomas and phaeochromocytomas. Recessive germline mutations in SDHB have recently been associated with complex II deficiency and leukodystrophy in one patient. METHODS AND RESULTS We present the clinical and molecular investigations of the first patient with biochemical evidence of a severe isolated complex II deficiency due to compound heterozygous SDHD gene mutations. The patient presented with early progressive encephalomyopathy due to compound heterozygous p.E69 K and p.*164Lext*3 SDHD mutations. Native polyacrylamide gel electrophoresis and western blotting demonstrated an impaired complex II assembly. Complementation of a patient cell line additionally supported the pathogenicity of the novel identified mutations in SDHD. CONCLUSIONS This report describes the first case of isolated complex II deficiency due to recessive SDHD germline mutations. We therefore recommend screening for all SDH genes in isolated complex II deficiencies. It further emphasises the importance of appropriate genetic counselling to the family with regard to SDHD mutations and their role in tumorigenesis.

Phosphate release kinetics in calcareous grassland and forest soils in response to H+ addition

Phosphate release kinetics in soils are of global interest because sustainable plant nutrition with phosphate will be a major concern in the future. Dissolution of phosphate-containing minerals induced by a changing rhizosphere equilibrium through proton input is one important mechanism that releases phosphate into bioavailable forms. Our objectives were (i) to determine phosphate release kinetics during H+ addition in calcareous soils of the Schwäbische Alb, Germany, and to assess the influence of (ii) land-use type (grassland vs. forest) and (iii) management intensity on reactive phosphate pools and phosphate release rate constants during H+ addition. Phosphate release kinetics were characterized by a large fast-reacting phosphatepool, which could be attributed to poorly-crystalline calcium phosphates, and a small slow-reacting phosphate pool probably originating from carbonate-bearing hydroxylapatite. Both reactive phosphate pools—as well as total phosphate concentrations (TP) in soil—were greater in grassland than in forest soils. In organically fertilized grassland soils, concentrations of released phosphate were higher than in unfertilized soils, likely because organic fertilizers contain poorly-crystalline phosphate compounds which are further converted into sparingly soluble phosphate forms. Because of an enriched slow-reacting phosphate pool, mown pastures were characterized by a more continuous slow phosphate release reaction in contrast to clear biphasic phosphate release patterns in meadows. Consequently, managing phosphate release kinetics via management measures is a valuable tool to evaluate longer-term P availability in soil in the context of finite rock phosphate reserves on earth.

Congenital proprotein convertase 1/3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort

BACKGROUND & AIMS Proprotein convertase 1/3 (PC1/3) deficiency, an autosomal-recessive disorder caused by rare mutations in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 also have been associated with obesity in heterozygotes in several population-based studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency. METHODS We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1. We measured enzymatic activity of recombinant PC1/3 proteins. RESULTS We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears. CONCLUSIONS In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1, failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in the processing of one or more enteric hormones that are required for nutrient absorption.

Loss of Cdx2 Expression in Primary Tumors and Lymph Node Metastases is Specific for Mismatch Repair-Deficiency in Colorectal Cancer

Background: Approximately 20% of all colorectal cancers are hypothesized to arise from the "serrated pathway" characterized by mutation in BRAF, high-level CpG Island Methylator Phenotype, and microsatellite instability/mismatch repair (MMR)-deficiency. MMR-deficient cancers show frequent losses of Cdx2, a homeodomain transcription factor. Here, we determine the predictive value of Cdx2 expression for MMR-deficiency and investigate changes in expression between primary cancers and matched lymph node metastases. Methods: Immunohistochemistry for Cdx2, Mlh1, Msh2, Msh6, and Pms2 was performed on whole tissue sections from 201 patients with primary colorectal cancer and 59 cases of matched lymph node metastases. Receiver operating characteristic curve analysis and Area under the Curve (AUC) were investigated; association of Cdx2 with clinicopathological features and patient survival was carried out. Results: Loss of Cdx2 expression was associated with higher tumor grade (p = 0.0002), advanced pT (p = 0.0166), and perineural invasion (p = 0.0228). Cdx2 loss was an unfavorable prognostic factor in univariate (p = 0.0145) and multivariate [p = 0.0427; HR (95% CI): 0.58 (0.34-0.98)] analysis. The accuracy (AUC) for discriminating MMR-proficient and - deficient cancers was 87% [OR (95% CI): 0.96 (0.95-0.98); p < 0.0001]. Specificity and negative predictive value for MMR-deficiency was 99.1 and 96.3%. One hundred and seventy-four patients had MMR-proficient cancers, of which 60 (34.5%) showed Cdx2 loss. Cdx2 loss in metastases was related to MMR-deficiency (p < 0.0001). There was no difference in expression between primary tumors and matched metastases. Conclusion: Loss of Cdx2 is a sensitive and specific predictor of MMR-deficiency, but is not limited to these tumors, suggesting that events "upstream" of the development of microsatellite instability may impact Cdx2 expression.

Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

BIOCHEMICAL CHARACTERIZATION AND GENETIC MAPPING OF WILD TYPE AND MUTANT GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ALLELES IN CHINESE HAMSTER OVARY CELLS

A UV-induced mutation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) was characterized in the CHO clone A24. The asymmetric 4-banded zymogram and an in vitro GAPD activity equal to that of wild type cells were not consistent with models of a mutant heterozygote producing equal amounts of wild type and either catalytically active or inactive mutant subunits that interacted randomly. Cumulative evidence indicated that the site of the mutation was the GAPD structural locus expressed in CHO wild type cells, and that the mutant allele coded for a subunit that differed from the wild type subunit in stability and kinetics. The evidence included the appearance of a fifth band, the putative mutant homotetramer, after addition of the substrate glyceraldehyde-3-phosphate (GAP) to the gel matrix; dilution experiments indicating stability differences between the subunits; experiments with subsaturating levels of GAP indicating differences in affinity for the substrate; GAPD zymograms of A24 x mouse hybrids that were consistent with the presence of two distinct A24 subunits; independent segregation of A24 wild type and mutant electrophoretic bands from the hybrids, which was inconsistent with models of mutation of a locus involved in posttranslational modification; the mapping of both wild type and mutant forms of GAPD to chromosome 8; and the failure to detect any evidence of posttranslational modification (of other A24 isozymes, or through mixing of homogenates of A24 and mouse).^ The extent of skewing of the zymogram toward the wild type band, and the unreduced in vitro activity were inconsistent with models based solely on differences in activity of the two subunits. Comparison of wild type homotetramer bands in wild type cells and A24 suggested the latter had a preponderance of wild type subunits over mutant subunits, and had more GAPD tetramers than did CHO controls.^ Two CHO linkages, GAPD-triose phosphate isomerase, and acid phosphatase 2-adenosine deaminase were reported provisionally, and several others were confirmed. ^

Re-emerging Vitamin D deficiency epidemic and its implications on the public health in the US

Vitamin D is essential in maintaining the bone health and Calcium homeostasis in the body. These actions are mediated through the Vitamin D receptors (VDR) present in cells through which the activated vitamin D acts [1]. In the past, it was known that these receptors existed in the intestine and bone cell. However, recent discovery of VDR in other tissues as well, has broadened the action of Vitamin D and increased its adequate intake [1].^ In the past, Vitamin D deficiency was most common among institutionalized, elderly patients and children and thought to be extinct in the healthy population. However, recent evidence has shown that, prevalence of vitamin D deficiency is increasing into an epidemic status in the overall population of the United States, including the healthy individuals [2-3]. The increased daily-recommended requirement and other multiple factors are responsible for the re-emergence of this epidemic [4-5]. Some of these factors could be used to control the epidemic. Studies have also shown the association between vitamin D deficiency and increased risk for developing chronic diseases such as diabetes, hypertension, multiple sclerosis, arthritis, and some fatal cancers like prostate, colon and breast cancers [1, 4, 6-14]. This issue results in increased disease burden, morbidity and mortality in the community [15-20].^ Methods: The literature search was conducted using the University of Texas Health Science Center at Houston (UTHSC) and University of Texas Southwestern Medical Center (UTSW) online library. The key search terms used are “vitamin D deficiency And prevalence Or epidemiology”, “vitamin D deficiency And implication And public health” using PubMed and Mesh database and “vitamin D deficiency” using systematic reviews. The search is limited to Humans and the English language. The articles considered for the review are limited to Healthy US population to avoid health conditions that predispose the population to vitamin D deficiency. Only US population is considered to narrow down the study.^ Results: There is an increased prevalence of low levels of Vitamin D levels below the normal range in the US population regardless of age and health status. Vitamin D deficiency is also associated with increased risk of chronic illnesses and fatal cancers.^ Conclusion: This increased prevalence and the association of the deficiency with increased all-cause mortality has increased the economic burden and compromised the quality of life among the population. This necessitates the health care providers to routinely screen their patients for the Vitamin D status and counsel them to avoid the harmful effects of the Vitamin D deficiency. ^