Evaluation Of Red Cell And Reticulocyte Parameters As Indicative Of Iron Deficiency In Patients With Anemia Of Chronic Disease.

The purpose of this study was to evaluate the effectiveness of mature red cell and reticulocyte parameters under three conditions: iron deficiency anemia, anemia of chronic disease, and anemia of chronic disease associated with absolute iron deficiency. Peripheral blood cells from 117 adult patients with anemia were classified according to iron status, and inflammatory activity, and the results of a hemoglobinopathy investigation as: iron deficiency anemia (n=42), anemia of chronic disease (n=28), anemia of chronic disease associated with iron deficiency anemia (n=22), and heterozygous β thalassemia (n=25). The percentage of microcytic red cells, hypochromic red cells, and levels of hemoglobin content in both reticulocytes and mature red cells were determined. Receiver operating characteristic analysis was used to evaluate the accuracy of the parameters in differentiating between the different types of anemia. There was no significant difference between the iron deficient group and anemia of chronic disease associated with absolute iron deficiency in respect to any parameter. The percentage of hypochromic red cells was the best parameter to discriminate anemia of chronic disease with and without absolute iron deficiency (area under curve=0.785; 95% confidence interval: 0.661-0.909, with sensitivity of 72.7%, and specificity of 70.4%; cut-off value 1.8%). The formula microcytic red cells minus hypochromic red cells was very accurate in differentiating iron deficiency anemia and heterozygous β thalassemia (area under curve=0.977; 95% confidence interval: 0.950-1.005; with sensitivity of 96.2%, and specificity of 92.7%; cut-off value 13.8). The indices related to red cells and reticulocytes have a moderate performance in identifying absolute iron deficiency in patients with anemia of chronic disease.

Metal Ions Bound To The Human Milk Immunoglobulin A: Metalloproteomic Approach.

The presence of calcium, iron, and zinc bound to human milk secretory IgA (sIgA) was investigated. The sIgA components were first separated by two-dimensional polyacrylamide gel electrophoresis and then identified by electrospray ionization-tandem mass spectrometry (ESI MS MS). The metal ions were detected by flame atomic absorption spectrometry after acid mineralization of the spots. The results showed eight protein spots corresponding to the IgA heavy chain constant region. Another spot was identified as the transmembrane secretory component. Calcium was bound to both the transmembrane component and the heavy chain constant region, while zinc was bound to the heavy chain constant region and iron was not bound with the identified proteins. The association of a metal ion with a protein is important for a number of reasons, and therefore, the findings of the present study may lead to a better understanding of the mechanisms of action and of additional roles that sIgA and its components play in human milk.

Nek5 Interacts With Mitochondrial Proteins And Interferes Negatively In Mitochondrial Mediated Cell Death And Respiration.

Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2μM). Nek5 silenced cells as well as cells expressing a kinase dead version of Nek5, displayed an increase in ROS formation after 4h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of hNek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro. Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells.

Characterization Of A Protein-protein Interaction Network Of The Cbl-interacting Protein Kinase 8 From Sugarcane.

Plants are sessile organisms and have evolved to tolerate a constantly changing environment. After the onset of different stress conditions, calcineurin B-like (CBL) proteins can sense calcium signals and activate CBL-interacting protein kinase (CIPK) proteins, which can phosphorylate downstream proteins to reestablish plant homeostasis. Previous studies in the bioenergy crop sugarcane showed that the ScCIPK8 gene is induced by drought stress and is also related to sucrose content. Here, we have characterized the protein-protein interactions of ScCIPK8 with six CBL proteins (ScCBL1, ScCBL2, ScCBL3, ScCBL6, ScCBL9, and ScCBL10). Yeast two-hybrid assays showed that ScCIPK8 interacts with ScCBL1, ScCBL3, and ScCBL6. Bimolecular fluorescence complementation assays confirmed in planta the interactions that were observed in yeast cells. These findings give insights on the regulatory networks related to sugar accumulation and drought stress responses in sugarcane.

Chasing Cardiac Physiology And Pathology Down The Camkii Cascade.

Calcium dynamics is central in cardiac physiology, as the key event leading to the excitation-contraction coupling (ECC) and relaxation processes. The primary function of Ca(2+) in the heart is the control of mechanical activity developed by the myofibril contractile apparatus. This key role of Ca(2+) signaling explains the subtle and critical control of important events of ECC and relaxation, such Ca(2+) influx and SR Ca(2+) release and uptake. The multifunctional Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a signaling molecule that regulates a diverse array of proteins involved not only in ECC and relaxation, but also in cell death, transcriptional activation of hypertrophy, inflammation and arrhythmias. CaMKII activity is triggered by an increase in intracellular Ca(2+) levels. This activity can be sustained, creating molecular memory after the decline in Ca(2+) concentration, by autophosphorylation of the enzyme, as well as by oxidation, glycosylation and nitrosylation at different sites of the regulatory domain of the kinase. CaMKII activity is enhanced in several cardiac diseases, altering the signaling pathways by which CaMKII regulates the different fundamental proteins involved in functional and transcriptional cardiac processes. Dysregulation of these pathways constitutes a central mechanism of various cardiac disease phenomena, like apoptosis and necrosis during ischemia/reperfusion injury, digitalis exposure, post-acidosis and heart failure arrhythmias, or cardiac hypertrophy. Here we summarize significant aspects of the molecular physiology of CaMKII and provide a conceptual framework for understanding the role of the CaMKII cascade on Ca(2+) regulation and dysregulation in cardiac health and disease.

Micrornas And Drought Responses In Sugarcane.

There is a growing demand for renewable energy, and sugarcane is a promising bioenergy crop. In Brazil, the largest sugarcane producer in the world, sugarcane plantations are expanding into areas where severe droughts are common. Recent evidence has highlighted the role of miRNAs in regulating drought responses in several species, including sugarcane. This review summarizes the data from miRNA expression profiles observed in a wide array of experimental conditions using different sugarcane cultivars that differ in their tolerance to drought. We uncovered a complex regulation of sugarcane miRNAs in response to drought and discussed these data with the miRNA profiles observed in other plant species. The predicted miRNA targets revealed different transcription factors, proteins involved in tolerance to oxidative stress, cell modification, as well as hormone signaling. Some of these proteins might regulate sugarcane responses to drought, such as reduction of internode growth and shoot branching and increased leaf senescence. A better understanding on the regulatory network from miRNAs and their targets under drought stress has a great potential to contribute to sugarcane improvement, either as molecular markers as well as by using biotechnological approaches.

Reduction Of Oxidative Damage And Inflammatory Response In The Diaphragm Muscle Of Mdx Mice Using Iron Chelator Deferoxamine.

Oxidative stress and inflammatory processes strongly contribute to pathogenesis in Duchenne muscular dystrophy (DMD). Based on evidence that excess iron may increase oxidative stress and contribute to the inflammatory response, we investigated whether deferoxamine (DFX), a potent iron chelating agent, reduces oxidative stress and inflammation in the diaphragm (DIA) muscle of mdx mice (an experimental model of DMD). Fourteen-day-old mdx mice received daily intraperitoneal injections of DFX at a dose of 150 mg/kg body weight, diluted in saline, for 14 days. C57BL/10 and control mdx mice received daily intraperitoneal injections of saline only, for 14 days. Grip strength was evaluated as a functional measure, and blood samples were collected for biochemical assessment of muscle fiber degeneration. In addition, the DIA muscle was removed and processed for histopathology and Western blotting analysis. In mdx mice, DFX reduced muscle damage and loss of muscle strength. DFX treatment also resulted in a significant reduction of dystrophic inflammatory processes, as indicated by decreases in the inflammatory area and in NF-κB levels. DFX significantly decreased oxidative damage, as shown by lower levels of 4-hydroxynonenal and a reduction in dihydroethidium staining in the DIA muscle of mdx mice. The results of the present study suggest that DFX may be useful in therapeutic strategies to ameliorate dystrophic muscle pathology, possibly via mechanisms involving oxidative and inflammatory pathways.

Saturated Fatty Acids Modulate Autophagy's Proteins In The Hypothalamus.

Autophagy is an important process that regulates cellular homeostasis by degrading dysfunctional proteins, organelles and lipids. In this study, the hypothesis that obesity could lead to impairment in hypothalamic autophagy in mice was evaluated by examining the hypothalamic distribution and content of autophagic proteins in animal with obesity induced by 8 or 16 weeks high fat diet to induce obesity and in response to intracerebroventricular injections of palmitic acid. The results showed that chronic exposure to a high fat diet leads to an increased expression of inflammatory markers and downregulation of autophagic proteins. In obese mice, autophagic induction leads to the downregulation of proteins, such as JNK and Bax, which are involved in the stress pathways. In neuron cell- line, palmitate has a direct effect on autophagy even without inflammatory activity. Understanding the cellular and molecular bases of overnutrition is essential for identifying new diagnostic and therapeutic targets for obesity.

Abnormally increased iron concentration in basal ganglia in Shy-Drager syndrome MR imaging and autonomic study: RM imagem e estudo autonômico

Report of an early case of Shy-Drager syndrome in a 67 year-old woman patient. Autonomic failure was diagnosed by functional evaluation as well as laboratory tests. MR imaging disclosed a prominent putamina hypodensity in T2-weighted images at high field strength due to iron increased depositing in this basal ganglia. MR imaging evidences confirm Shy-Drager syndrome diagnosis, and contributes for differential diagnosis of idiopathic hypotension (pure autonomic failure) in special in SDS early cases.

Association of polymorphisms at the ADIPOR1 regulatory region with type 2 diabetes and body mass index in a Brazilian population with European or African ancestry

Association studies between ADIPOR1 genetic variants and predisposition to type 2 diabetes (DM2) have provided contradictory results. We determined if two single nucleotide polymorphisms (SNP c.-8503G>A and SNP c.10225C>G) in regulatory regions of ADIPOR1 in 567 Brazilian individuals of European (EA; N = 443) or African (AfA; N = 124) ancestry from rural (quilombo remnants; N = 439) and urban (N = 567) areas. We detected a significant effect of ethnicity on the distribution of the allelic frequencies of both SNPs in these populations (EA: -8503A = 0.27; AfA: -8503A = 0.16; P = 0.001 and EA: 10225G = 0.35; AfA: 10225G = 0.51; P < 0.001). Neither of the polymorphisms were associated with DM2 in the case-control study in EA (SNP c.-8503G>A: DM2 group -8503A = 0.26; control group -8503A = 0.30; P = 0.14/SNP 10225C>G: DM2 group 10225G = 0.37; control group 10225G = 0.32; P = 0.40) and AfA populations (SNP c.-8503G>A: DM2 group -8503A = 0.16; control group -8503A = 0.15; P = 0.34/SNP 10225C>G: DM2 group 10225G = 0.51; control group 10225G = 0.52; P = 0.50). Similarly, none of the polymorphisms were associated with metabolic/anthropometric risk factors for DM2 in any of the three populations, except for HDL cholesterol, which was significantly higher in AfA heterozygotes (GC = 53.75 ± 17.26 mg/dL) than in homozygotes. We conclude that ADIPOR1 polymorphisms are unlikely to be major risk factors for DM2 or for metabolic/anthropometric measurements that represent risk factors for DM2 in populations of European and African ancestries.

Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues

OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

Biological activity of metal-edds (ethylenediaminedisuccinate) complexes in K562 and PBMC cells

The effect of S,S-ethylenediaminedisuccinic acid (edds) on the quenching of metal-catalyzed (metal = Mn, Fe, Co, Ni, Cu, Zn) oxidation of ascorbic acid was tested in vitro via oxidation of the fluorescent probe 1,2,3-dihydrorhodamine dihydrochloride. The pro-oxidant activity of iron was not fully suppressed, even at a four-fold molar excess of the ligand. The effect of serum on the toxicity to peripheral blood mononuclear cells (PBMC) and K562 cells was investigated. The cytotoxic effect of Fe-edds was abrogated in the presence of Trolox or serum proteins. The probable pathways of cell toxicity were investigated through blocking of the monocarboxylate transporters (MCT) in association with cell cycle studies by flow cytometry. Cells treated with metal complexes and alpha-cyano-4-hydroxycinnamic acid, a known MCT inhibitor, showed recovery of viability, suggesting that MCT proteins may be involved in the internalization of metal-edds complexes. The free acid induced cell cycle arrest in G0/G1 (PBMC) and S (K562) phases, suggesting direct DNA damage or interference in DNA replication.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Modified silicas covalently bounded to 5,10,15,20-tetrakis(2-hydroxy-5-nitrophenyl)porphyrinato iron(III): synthesis, spectroscopic and EPR characterization. Catalytic studies

In this work we have studied cyclooctene epoxidation with PhIO, using a new iron porphyrin, 5,10,15,20-tetrakis(2-hydroxy-5-nitrophenyl)porphyrinato iron(III), supported on silica matrices via eletrostatic interaction and / or covalent bonds as catalyst. These catalysts were obtained and immobilized on the solid supports propyltrimethylammonium silica (SiN+); propyltrimethylammonium and propylimidazole silica [SiN+(IPG)] and chloropropylsilica (CPS) via elestrostatic interactions and covalent binding. Characterization of the supported catalysts by UV-Vis spectroscopy and EPR (Electron paramagnetic resonance) indicated the presence of a mixture of FeII and FeIII species in all of the three obtained catalysts. In the case of (Z)-cyclooctene epoxidation by PhIO the yields observed for cis-epoxycyclooctane were satisfactory for the reactions catalyzed by the three materials (ranging from 68% to 85%). Such results indicate that immobilization of metalloporphyrins onto solid supports via groups localized on the ortho positions of their mesophenyl rings can lead to efficient catalysts for epoxidation reactions. The catalyst 1-CPS is less active than 1-SiN and 1-SiN(IPG), this argues in favour of the immobilization of this metalloporphyrin onto solids via electrostatic interactions, which is easier to achieve and results in more active oxidation catalysts. Interestingly, the activity of the supported catalysts remained the same even after three successive recyclings; therefore, they are stable under the oxidizing conditions.