Metal binding proteins or metallo-proteins are important for the stability of the protein and also serve as co-factors in various functions like controlling metabolism, regulating signal transport, and metal homeostasis. In structural genomics, prediction of metal binding proteins help in the selection of suitable growth medium for overexpression’s studies and also help in obtaining the functional protein. Computational prediction using machine learning approach has been widely used in various fields of bioinformatics based on the fact all the information contains in amino acid sequence. In this study, random forest machine learning prediction systems were deployed with simplified amino acid for prediction of individual major metal ion binding sites like copper, calcium, cobalt, iron, magnesium, manganese, nickel, and zinc.
Amino Acid Sequence* ; Binding Sites* ; Calcium ; Carrier Proteins ; Cobalt ; Computational Biology ; Copper ; Forests* ; Genomics ; Homeostasis ; Iron ; Machine Learning ; Magnesium ; Manganese ; Metabolism ; Nickel ; Zinc

OBJECTIVETo assess the association of thyroperoxidase (TPO) gene polymorphisms with dyshormonogenesis in congenital hypothyroidism (CH).

METHODSThe 17 exons and flanking introns of the TPO gene from 30 randomly selected samples were sequenced for the selection of single nucleotide polymorphisms (SNPs). In 136 patients with dyshormonogenetic CH and 141 healthy controls from the same region, the selected SNPs were genotyped by polymerase chain reaction (PCR) and direct sequencing or PCR-restriction fragment length polymorphism (RFLP).

RESULTSSix SNPs (rs9678281, rs376413622, rs1126797, rs4927611, rs732609 and rs1126799) were selected to determine the genotype for each sample. Among these, rs4927611 and rs732609 showed a significant difference between the two groups in both allelic and genotypic frequencies. With a recessive model of inheritance, rs732609 CC (OR=0.484, 95%CI: 0.253-0.927, P=0.04) and rs4927611 TT (OR=0.32, 95%CI: 0.112-0.915, P=0.047) were greater in the patients.

CONCLUSIONrs4927611 and rs732609 may be associated with dyshormonogenetic CH. rs4927611 TT and rs732609 CC are genotypes associated with potential risk for the disease.

Alleles ; Autoantigens ; genetics ; Base Sequence ; Child, Preschool ; Congenital Hypothyroidism ; blood ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Humans ; Infant ; Infant, Newborn ; Iodide Peroxidase ; genetics ; Iron-Binding Proteins ; genetics ; Linkage Disequilibrium ; Male ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Risk Factors ; Thyrotropin ; blood ; Thyroxine ; blood

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of Ca2+, Mg2+, K+, and HCO3 - in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.
ATP-Binding Cassette Transporters/*chemistry/*genetics/metabolism ; Adenosine Triphosphate/metabolism ; Amino Acid Sequence ; Animals ; Calcium/metabolism ; *Cloning, Molecular ; Cryptosporidiosis/parasitology ; Cryptosporidium/chemistry/genetics/*metabolism ; Humans ; Iron/metabolism ; Mice ; Molecular Sequence Data ; Protein Structure, Tertiary ; Protozoan Proteins/*chemistry/*genetics/metabolism ; Sequence Alignment

DNA primase catalyzes de novo synthesis of a short RNA primer that is further extended by replicative DNA polymerases during initiation of DNA replication. The eukaryotic primase is a heterodimeric enzyme comprising a catalytic subunit Pri1 and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pri1 and mediating interaction between Pri1 and DNA polymerase α for transition from RNA synthesis to DNA elongation. All eukaryotic Pri2 proteins contain a conserved C-terminal iron-sulfur (Fe-S) cluster-binding domain that is critical for primase catalytic activity in vitro. Here we show that mutations at conserved cysteine ligands for the Pri2 Fe-S cluster markedly decrease the protein stability, thereby causing S phase arrest at the restrictive temperature. Furthermore, Pri2 cysteine mutants are defective in loading of the entire DNA pol α-primase complex onto early replication origins resulting in defective initiation. Importantly, assembly of the Fe-S cluster in Pri2 is impaired not only by mutations at the conserved cysteine ligands but also by increased oxidative stress in the sod1Δ mutant lacking the Cu/Zn superoxide dismutase. Together these findings highlight the critical role of Pri2's Fe-S cluster domain in replication initiation in vivo and suggest a molecular basis for how DNA replication can be influenced by changes in cellular redox state.
Amino Acid Sequence ; Cell Cycle ; Cell Proliferation ; Chromatin Immunoprecipitation ; Cysteine ; genetics ; metabolism ; DNA Primase ; genetics ; metabolism ; DNA Replication ; DNA, Fungal ; genetics ; DNA-Directed DNA Polymerase ; metabolism ; Immunoblotting ; Immunoprecipitation ; Iron ; metabolism ; Iron-Sulfur Proteins ; metabolism ; Molecular Sequence Data ; Mutation ; genetics ; Oxidative Stress ; Protein Binding ; Saccharomyces cerevisiae ; genetics ; growth & development ; metabolism ; Sequence Homology, Amino Acid ; Sulfur ; metabolism

BACKGROUND: To determine the thyroid ultrasound findings in association with anti-TPO positivity among patients with diffuse goiter.
DESIGN AND METHODS: We performed a cross-sectional study on patients with diffuse goiter seen at Makati Medical Center out-patient Endocrine clinics from October 1, 2011 to October 1, 2012. Patients with anti-TPO (thyroid peroxidase) above 100 pmol/L were considered anti-TPO positive and below this level were considered negative. After excluding patients with other possible causes of thyroiditis, thyroid ultrasound of anti-TPO positive and anti-TPO negative patients were reviewed and compared based on size, echogenicity, echopattern and vascularity of the thyroid parenchyma.
RESULTS: In 94 patients who qualified for the study, 43.6% were anti-TPO positive. A higher proportion of anti-TPO positive was seen among females compared to males by almost twofold (49.7% vs 25%, p0.05). Stratified according to age group for female patients, anti-TPO positivity is relatively higher among 31-50 years old (51.1%, p =0.753). Among male, anti-TPO positivity is present in all 18-2 years old which is significantly higher compared to other age group (p <0.01). Based on thyroid ultrasound findings, those with positive anti-TPO has
larger thyroid size in all measurement parameters (p= 0.0053). Among anti-TPO positive patients, frequent ultrasound findings were: hypoechoic (79% vs. 21%, p 0.001); heterogenous parenchyma (71% vs. 29%, p 0.001) and increased vascularity (93% vs. 7%, p 0.001). Of note is the absence of homogenous prenchyma fnding among anti-TPO positive. All 23 (100%) patients who showed combined findings of hypoechoic, heterogenous parenchyma and increased vascularity were anti-TPO positive.
CONCLUSION: Thyroid ultrasound findings that are found frequently among anti-TPO positive are increased thyroid size, parenchyma that are hypoechoiec and heterogenous and increased vascularity. Homogenous echotexture was not seen among anti-TPO positive. The combined sonographic characteristics of hypoechoic, heterogenous pattern and increased vascularity are highly suggestive of presence of anti-TPO (100%).

Human ; Male ; Female ; Aged ; Middle Aged ; Adult ; Tpo Protein, Human ; Iodide Peroxidase ; Iron-binding Proteins ; Autoantigens ; Thyroiditis ; Goiter

OBJECTIVETo study relationships between serum ferritin and bone metabolism in patients with hip fragility fractures.

METHODSThis cross-sectional study included 76 postmenopausal women with hip fracture from Feburary 2011 to June 2012. The mean age of the women was (73 ± 10) years (range, 55-93 years) and the mean duration of menstruation was (22 ± 10)years (range, 5-50 years). Serum concentrations of ferritin, transferrin, alkaline phosphatase (ALP), amino-terminal extension peptide of type I collagen (P1NP), C-terminal telopeptides of type I collagen (β-CTX)and femoral and lumbar bone mineral density by dual-energy X-ray absorptiometry were measured. Bone metabolism was compared between normal and elevated ferritin groups with t-test, Pearson linear, partial correlation and multiple regression analysis examined associations between iron- and bone-related markers.

RESULTSSerum ferritin concentration raised to (230 ± 146)µg/L, transferrin concentration reduced to (1.89 ± 0.33)g/L. P1NP concentration raised to (61 ± 32) ng/L when the concentration of serum ALP and β-CTX were in the normal range. T-scores for bone mineral density in the femoral neck (-2.0 ± 1.1) and lumbar (-2.1 ± 1.2) were below the normal ranges(-1.0-1.0). The subjects were divided into two groups according to serum ferritin concentration, normal group(serum ferritin concentration ≤ 150 µg/L, n = 25) and elevated group(serum ferritin concentration > 150 µg/L, n = 51). Patients of elevated group had lower bone mineral density in femoral neck and lumbar than normal group(t = 3.13,2.89, P < 0.01), and higher P1NP, β-CTX concentration (t = -2.38, -3.59, P < 0.05) . In partial correlation analysis adjusted for confounders, serum ferritin concentration was correlated negatively with bone mineral density in both femoral neck and lumbar (r = -0.335,-0.295, P < 0.05), and positively with P1NP and β-CTX (r = 0.467,0.414, P < 0.05), but not correlated with ALP (r = 0.188, P > 0.05). Transferrin concentration tended to be correlated positively with bone mineral density in both femoral neck and lumbar (r = 0.444, 0.262, P < 0.05) and negatively with ALP, P1NP and β-CTX(r = -0.326,-0.285,-0.278, P < 0.05).

CONCLUSIONSIron overload has a high prevalence in postmenopausal women with fragility fracture. Increased iron stores, which might lead to bone loss and lower bone mineral density by enhancing the activity of bone turnover, could be an independent factor to take effects on bone metabolism on postmenopausal women.

Aged ; Aged, 80 and over ; Bone Density ; Bone Remodeling ; Collagen Type I ; blood ; Cross-Sectional Studies ; Female ; Hip Fractures ; metabolism ; Humans ; Iron Overload ; Iron-Binding Proteins ; metabolism ; Middle Aged ; Osteoporosis, Postmenopausal ; metabolism ; Postmenopause ; Retrospective Studies

This study examined the effects of a brown rice vegetarian diet and outdoor walking exercise program on body composition and blood lipid parameters in collegians. The mean age of respondents was 21.8 yrs (males) and 21.7 yrs (females). During the ten-day program, the respondents lived in a dormitory and had three meals. The respondents exercised one hour in the morning (6:20~7:20 am) and attended one and a half hour evening lecture (7:00~8:30 pm) everyday. The brown rice vegetarian diet consisted of brown rice, whole grain bread, beans, fresh vegetables, and fresh fruits contained 2043.2+/-112.7 kcal (97.3% of RNI), 66.7 g protein (133.3% of RNI), 33.6 g dietary fiber (168.2% of RNI), vitamin A (194.2% of RNI), vitamin B1 (245.5% of RNI), vitamin B2 (225.1% of RNI), niacin (233.7% of RNI), vitamin B6 (277.1% of RNI), folic acid (128.4% of RNI), vitamin C (334.6% of RNI), iron (131.9% of RNI), zinc (112.4% of RNI) and calcium (60.3% of RNI). The results showed that there were significant increases in body weight (P<0.05) and BMI (P<0.05) in males and body weight (P<0.05) and lean body mass (P<0.01) in females. In addition, there were significant decreases in total cholesterol (P<0.001), LDL cholesterol (P<0.001), TG (P<0.05), and HDL-cholesterol (P<0.001) in males and total cholesterol (P<0.01) and LDL-cholesterol (P<0.01) in female. The ten day brown rice vegetarian diet rich in fiber and outdoor walking exercise program significantly increased body weight and decreased total cholesterol and LDL-cholesterol in collegians.
Ascorbic Acid ; Body Composition ; Body Weight ; Bread ; Calcium ; Edible Grain ; Cholesterol ; Cholesterol, LDL ; Surveys and Questionnaires ; Diet, Vegan ; Dietary Fiber ; Fabaceae ; Female ; Folic Acid ; Fruit ; GTP-Binding Proteins ; Humans ; Iron ; Male ; Meals ; Niacin ; Riboflavin ; Thiamine ; Vegetables ; Vitamin A ; Vitamin B 6 ; Walking ; Zinc

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression levels of the frataxin gene (FXN) due to expansion of triplet nucleotide GAA repeats in the first intron of FXN. FXN is a mitochondrial protein which plays an important role in the regulation of intracellular iron trafficking, biogenesis of iron-sulfur cluster and heme, and removal of reactive oxygen species. Our previous work showed that tissue-specific expression of FXN in cerebellum and heart generates two novel isoforms. In order to find the isoforms in mouse tissues, we tried to obtain a polyclonal antibody against mouse Fxn with high specificity and sensitivity. Thus, the recombinant plasmid pET24(+)-mFxn was constructed to express his-tagged Fxn in BL21 (DE3) cells. The expressed protein is a mature form with 130 amino acids (aa, 14.38 kDa) without the N-terminal signal peptide (77 aa), purified on Ni-NTA column and further dialyzed with Centrifugal Filtration Device. The polyclonal antibody against Fxn was produced by immunizing rabbits with highly purified protein. The collected antiserums were preliminarily purified by precipitation with (NH4)2SO4. Western blotting analysis and cell immunofluorescence showed that the obtained antibody was able to detect both purified and endogenous Fxn. It also worked well in immunoprecipitation with mouse tissues. This is the first time, to our knowledge, to report that mouse Fxn was used as immunogen to generate antibody with high specificity and sensitivity. This work provides a powerful tool for our further research on mouse Fxn isoforms.
Animals ; Antibodies ; immunology ; metabolism ; Antibody Specificity ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Engineering ; methods ; Immunization ; Iron-Binding Proteins ; genetics ; immunology ; Mice ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

The elderly are a peculiar group in terms of health management, as they often present with non-specific complaints which are challenging to interpret and may not present with the usual clinical picture of a disease. Objective. The study aims to determine the prevalence of thyroid dysfunction among asymptomatic, elderly Filipinos seen at the Philippine General Hospital (PGH). Methodology. Subjects aged 60 years and older seeking out-patient medical consult for non-thyroidal illness at the PGH were recruited. Patients with known thyroid or pituitary disease, previous thyroid or pituitary surgery, intake of medications known to affect thyroid hormone levels and critical illness were excluded. Fasting blood sugar (FBS), lipid profile, free thyroxine (FT4), thyroid-stimulating hormone (TSH), and anti-thyroperoxidase (anti-TPO) levels were taken. Based on FT4 and TSH levels, subjects were classified as overt hypothyroid, subclinical hypothyroid, euthyroid, subclinical hyperthyroid, or overt hyperthyroid. Results. One hundred eighty subjects were recruited, of whom 152 (84%) were female. Hypertension was the most common comorbidity (58.33%), followed by diabetes (36.67%). One hundred sixty-two (90%) were euthyroid, 12 (6.7%) subclinical hypothyroid, 4 (2.22%) subclinical hyperthyroid, and two (1.11%) overtly hyperthyroid. No one was overtly hypothyroid. There was a trend toward increasing prevalence of diabetes, hypertension, low HDL, obesity and overall cardiovascular risk among those with subclinical hypothyroidism. Conclusion. Subclinical hypothyroidism was the most prevalent thyroid dysfunction among asymptomatic elderly included in the study.

Human ; Male ; Female ; Aged 80 And Over ; Aged ; Cardiovascular Diseases ; Diabetes Mellitus ; Hospitals, General ; Hypertension ; Hyperthyroidism ; Hypothyroidism ; Iodide Peroxidase ; Iron-binding Proteins ; Obesity ; Outpatients ; Pituitary Diseases ; Thyrotropin ; Thyroxine

Radioiodine is a routine therapy for differentiated thyroid cancers. Non-thyroid cancers can intake radioiodine after transfection of the human sodium iodide symporter (hNIS) gene. The human telomerase reverse transcriptase (hTERT) promoter, an excellent tumor-specific promoter, has potential value for targeted gene therapy of glioma. We used the hTERT promoter to drive the expression of the hNIS and human thyroid peroxidase (hTPO) gene as a primary step for testing the effects of radioiodine therapy on malignant glioma. The U87 and U251 cells were co-transfected with two adenoviral vectors, in which the hNIS gene had been coupled to the hTERT promoter and the hTPO gene had been coupled to the CMV promoter, respectively. Then, we performed Western blot, 125I intake and efflux assays, and clonogenic assay with cancer cells. We also did 99mTc tumor imaging of nude mice models. After co-transfection with Ad-hTERT-hNIS and Ad-CMV-hTPO, glioma cells showed the 125I intake almost 1.5 times higher than cells transfected with Ad-hTERT-hNIS alone. Western blots revealed bands of approximately 70 kDa and 110 kDa, consistent with the hNIS and hTPO proteins. In clonogenic assay, approximately 90% of co-transfected cells were killed, compared to 50% of control cells after incubated with 37 MBq of 131I. These results demonstrated that radioiodine therapy was effective in treating malignant glioma cell lines following induction of tumor-specific iodide intake by the hTERT promoter-directed hNIS expression in vitro. Co-transfected hNIS and hTPO genes can result in increased intake and longer retention of radioiodine. Nude mice harboring xenografts transfected with Ad-hTERT-NIS can take 99mTc scans.
Adenoviridae ; genetics ; Animals ; Autoantigens ; genetics ; metabolism ; Cell Line, Tumor ; Cell Survival ; Cytomegalovirus ; genetics ; Genetic Vectors ; Glioma ; diagnostic imaging ; genetics ; metabolism ; pathology ; Half-Life ; Humans ; Iodide Peroxidase ; genetics ; metabolism ; Iodine Radioisotopes ; metabolism ; Iron-Binding Proteins ; genetics ; metabolism ; Mice ; Mice, Nude ; Promoter Regions, Genetic ; Recombinant Proteins ; genetics ; metabolism ; Symporters ; genetics ; metabolism ; Technetium ; Telomerase ; genetics ; Tomography, Emission-Computed, Single-Photon ; Transfection