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PubMed2000

ETAAS method for the determination of Cd, Cr, Cu, Mn and Se in blood fractions and whole blood.

An electrothermal atomic absorption method (ETAAS) for the direct determination of trace elements (Cd, Cr, Cu, Mn, Se) both in blood fractions (erythrocytes, plasma and lymphocytes) and whole blood was developed. Zeeman background correction and graphite tubes with L'vov platforms were used. Samples were diluted with HNO3/Triton X-100 and pipetted directly into the graphite tube. Ashing, pretreatment and atomization steps were optimized carefully for the different fractions and elements applying different matrix modifiers for each element. For the lymphocyte fraction a multi-fold injection technique was applied. Low detection limits of the ETAAS method (Cd 0.13 microgram/L, Cr 0.11 microgram/L, Cu 0.52 microgram/L, Mn 0.13 microgram/L, Se 0.7 microgram/L of whole blood) combined with small quantities of sample necessary for analysis allow determination of trace elements in this matrix. Verification of possible differences in the trace element status of humans was performed with statistical significance (P < 0.05). In addition, a contribution to the determination of normal values of essential elements was achieved. The method was applied for determination of trace elements in human blood and blood fractions of two groups (n = 50) different in health status.

Erythrocytes↗

PubMed2005

The Impact of the hyperacid Ijen Crater Lake. Part II: A total diet study.

In Asembagus (East Java, Indonesia), surface water is contaminated with effluent from the hyperacid Ijen Crater Lake. In a previous study, we reported that food crops irrigated with this water had increased concentrations of various elements. Here, we present a total diet study for adults and 6-year-old children to determine if the mean daily intake of a broad range of elements is safe and adequate. For children, the intake of B, Mg, Mn and V is high with Hazard Quotients (HQ) of 1.1 (B), 1.4 (Mn) and 1.4 (V), respectively (no TDI is available for Mg). For Mn, the daily intake is high due to the consumption of locally produced rice. Drinking water is the main source of B, Mg and V. For adults, the intake of Mg, Mn and V is also high but HQ values are < or =0.7. For both children and adults, the intake of Ca, Zn and particularly Fe is below the RNI (Fe intake is 90% below the RNI for women). It is concluded the mean intake of elements is unbalanced and Fe deficiency is probably the most serious health problem. Toxic effects cannot be fully excluded since deficiency of essential elements such as Ca, Fe and Zn can increase the absorption and retention of various elements.

Adult↗

PubMed1996

Molecular cloning and characterization of a transcription factor for the C-type natriuretic peptide gene promoter.

Our previous studies on the promoter function of the human C-type natriuretic peptide (CNP) gene revealed the existence of two GC-rich cis elements essential for gene transcription in rat pituitary-derived GH3 cells. To isolate transcription factors that bind to those GC-rich elements, we screened a lambda ZAP cDNA library derived from GH3 cells by Southwestern screening. Several positive clones with specific binding abilities were obtained, and one was identical as TSC-22, a speculated transcriptional modulator stimulated by transforming growth factor beta (TGF-beta) of unknown function. TSC-22 significantly enhanced CNP promoter activity in GH3 cells. We further cloned a 1.8-kb full-length human TSC-22 cDNA from a fetal kidney cDNA library by a combination of polymerase chain reaction and the rapid amplification of the cDNA ends technique. In adults, human TSC-22 mRNA was highly expressed in brain, lung and heart. TSC-22 gene expression in GH3 and human aortic endothelial cells was stimulated by cytokines including TGF-beta, in correlation with the CNP mRNA increase. These results suggest that TSC-22 is a transcriptional regulator of the CNP gene and transmits signals from cytokines, such as TGF-beta, to CNP gene expression.

Adult↗

PubMed1988

Transmembrane signal transduction regulates gene expression in Dictyostelium discoideum.

cAMP regulates gene expression in Dictyostelium discoideum through the cell surface receptor and is therefore a transmembrane signal transduction event. We have now begun to examine the signal transduction pathway that transmits the cAMP-induced signal to the nucleus. The results presented here indicate that Ca2+ plays a crucial role. A comparison of the accumulation of UDPGP1 mRNA during development with the corresponding transcription rates revealed that this gene is regulated primarily at the level of transcription. To elucidate the factors involved in the regulation of the UDPGP1 gene we characterized its cis acting sequences. We constructed a series of deletions into the 5' flanking region of the UDPGP1 gene and analyzed the expression of the mutated DNA in transformants. A sequence element essential for the expression of the UDPGP1 gene is located between -500 bp and -288 bp from the transcription start site. This promoter element appears to be a short G + C-rich sequence positioned between -374 to -395 and coincides with a DNase I hypersensitive site.

Base Sequence↗

PubMed2005

Promoter activity of sea lamprey proopiocortin and proopiomelanotropin genes in AtT-20/D16v cells.

Adrenocorticotropic hormone (ACTH) and melanophore-stimulating hormone (MSH) are produced in the pars distalis and pars intermedia, respectively, throughout vertebrates. These hormones together with beta-endorphin are encoded on a single gene proopiomelanocortin (POMC) in gnathostomes, but in the sea lamprey, an agnathan, ACTH and MSH are encoded on two separate genes, proopiocortin (POC) and proopiomelanotropin (POM), respectively. Moreover, the nucleotide sequences of 5'-flanking regions of the POC and POM genes are significantly different from each other. To investigate the potential promoter activities of the POC and POM genes, we constructed promoter reporter plasmids by fusing the 5' flanking sequences (nucleotides -1151 to +31 and -2510 to +51, respectively) to a firefly luciferase gene. Transient transfection studies in AtT-20/D16v cells, which derived from a mouse pituitary tumor cell line, revealed that the 5'-flanking sequence of the POC gene did not exhibit promoter activity, whereas that of the POM gene showed the activity at high levels nearly equivalent to SV40 promoter. Analysis of a series of the 5'-deleted reporter for the POM gene in the AtT-20/D16v cells demonstrated that the 422 bp 5'-flanking sequence was sufficient for promoter activity, while the sequence from -853 to -574 may contain negatively acting regulatory elements. Because the POC and POM genes are supposed to have differentiated from a common ancestor, during evolution, the POC gene may lack essential element(s) for expression in the AtT-20/D16v cells.

Adrenocorticotropic Hormone↗

PubMed1988

Purification and functional characterization of a cellular transcription factor that binds to an enhancer element within the adenovirus early EIIa promoter.

The adenovirus EIa-inducible early EIIa (EIIaE) promoter is comprised of several sequence elements essential for constitutive and induced expression. We report here the purification of the host-cell factor that interacts with the major upstream element of this promoter, extending between positions -90 and -70 with respect to the main EIIaE cap site and exhibiting enhancer properties. The purified factor, which corresponds to a 40- to 43-kDa polypeptide, specifically binds to its recognition site and stimulates EIIaE promoter activity when added to an in vitro transcription system, reconstituted from purified factors and RNA polymerase. The implication of this factor in the control of the other adenovirus early genes is discussed.

Adenovirus Early Proteins↗

PubMed2004

Identification of a functional core in the RNA component of RNase MRP of budding yeasts.

RNase MRP is an endonuclease participating in ribosomal RNA processing. It consists of one RNA and at least nine protein subunits. Using oligonucleotide-directed mutagenesis, we analyzed the functional role of five of the hairpins in the secondary structure of the RNA subunit of Saccharomyces cerevisiae RNase MRP. Deletion of an entire hairpin was either lethal or resulted in very poor growth. However, peripheral portions constituting up to 70% of a hairpin could be deleted without effects on cell growth rate or processing of rRNA. To determine whether these hairpins perform redundant functions, we analyzed mutants combining four or five benign hairpin deletions. Simultaneous removal of four of these hairpin segments had no detectable effect. Removing five created a temperature- and cold-sensitive enzyme, but these deficiencies could be partially overcome by a mutation in one of the RNase MRP protein subunits, or by increasing the copy number of several of the protein subunit genes. These observations suggest that the peripheral elements of the RNA hairpins contain no structures or sequences required for substrate recognition, catalysis or binding of protein subunits. Thus, the functionally essential elements of the RNase MRP RNA appear to be concentrated in the core of the subunit.

Base Sequence↗

PubMed2003

Temperature sensing by the dsrA promoter.

Synthesis of the small regulatory RNA DsrA is under temperature control. The minimal dsrA promoter of 36 bp contains sufficient information to ensure such regulation. In vivo, we have analyzed the critical elements responsible for the temperature control of dsrA by using a collection of chimeric promoters combining various elements of the dsrA promoter and the lacUV5 promoter, which does not respond to temperature. Our results favor an RNA polymerase-DNA interaction model instead of a trans-acting factor for temperature regulation. While all of the elements of the dsrA promoter contribute to temperature-sensitive expression, the sequence of the -10 box and the spacer region are the essential elements for the thermal response of the dsrA promoter. The proper context for these promoter elements, including at least one of the flanking elements, the -35 region or the start site region, is also required. Point mutations demonstrate that the sequence of the -10 box imposes constraints on the length and the sequence of the spacer and/or its AT richness, even at low temperature. These results show a complex interdependence of different regions in the promoter for temperature regulation.

Bacterial Outer Membrane Proteins↗

PubMed2001

Permeability and the mechanism of transport of boric acid across the plasma membrane of Xenopus laevis oocytes.

Boron is an essential element for vascular plants and for diatoms, cyanobacteria, and a number of species of marine algal flagellates. Boron was recently established as an essential micronutrient for frogs (Xenopus laevis) and preliminary evidence suggests that it may be essential for all animals. The main form of B, which is available in the natural environment, is in the form of undissociated boric acid. The permeability coefficient and the mechanism of transport of boric acid, however, have not been experimentally determined across any animal membrane or cell. In the experiments described here, the permeability coefficient of boric acid in Xenopus oocytes was 1.5 x 10(-6) cm/s, which is very close with the permeability across liposomes made with phosphatidylcholine and cholesterol (the major lipids in the oocyte membrane). Moreover, we investigated the mechanism of boric acid movement across the membrane of Xenopus oocytes and we compared it with the transport across artificial liposomes. The transport of boric acid across Xenopus oocytes was not affected by inhibitors such as HgCl2, phloretin, or 4,4-diisothiocyanatostilbene-2,2'-D-sulfonic acid (DIDS). The kinetics of B uptake was linear with concentration changes, and the permeability remained the same at different external boric acid concentrations. These results suggest that B transport occurs via simple passive diffusion through the lipid bilayer in Xenopus oocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Narrow hybrid zone between two subspecies of big sagebrush (ARTEMISIA TRIDENTATA: Asteraceae). IX. Elemental uptake and niche separation.

The concentrations of selected elements and their biological absorption coefficients were determined for leaves from plants in native stands and reciprocal transplant gardens to determine whether niche differentiation occurs among the parental taxa and their hybrids in the big sagebrush hybrid zone in Utah. The bounded hybrid superiority model predicts such niche differentiation, while the ecologically neutral dynamic equilibrium model predicts complete niche overlap, at least in the vicinity of the hybrid zone. The concentrations of elements in the leaves of site-indigenous sagebrush and the biological absorption coefficients differed significantly between the subspecies and between either parental taxon and hybrids. Within reciprocal transplant gardens, both the elemental concentrations and the biological absorption coefficients differed among the gardens and taxa. Significant genotype-by-environment interactions were observed for several essential elements. Niche differentiation was evident as correspondence analyses ordinated the parental taxa and hybrids into separate groups even when raised in the same garden. These findings support the ecologically based bounded hybrid superiority model and suggest that the big sagebrush parental taxa and their hybrids have adapted to their respective unique habitats.

Journal Article↗

PubMed2002

Hepatic and renal concentrations of copper and other trace elements in hippopotami (Hippopotamus amphibius L) living in and adjacent to the Kafue and Luangwa Rivers in Zambia.

Hepatic and renal concentrations of the elements arsenic, cadmium, cobalt, copper, lead, manganese, mercury, molybdenum, selenium and zinc were studied in samples collected from hippopotami from the Kafue River in the Kafue National Park and the Luangwa River in the Southern Luangwa National Park in Zambia. There were no significant differences between trace element concentrations in the tissues of the hippopotami taken in the Kafue River and the Luangwa River. The concentrations of copper and other essential elements were similar to those reported in normal domestic and wild ruminants. Judging by the results obtained in this study, pollution from the mining activity around the Kafue River drainage area in the Copperbelt region has not led to any accumulation of elements in tissues of the hippopotami in the Kafue National Park. The trace element concentrations observed may serve as reference for similar future studies on hippopotami.

Animals↗

PubMed1987

Trace elements in the environment and in human organs. I. Methods and results.

The authors have determined by spectrophotometry the copper, zinc, lead, and cadmium content of the kidney, liver, lung, and adrenal derived from 1072 autopsies. The metallic content of food and drink was examined and the daily quantity consumed was stated by measuring and calculation. According to the results in Hungary the daily ingestion of these four microelements is small, as reflected by the metal content of the organs. An average 75% of the kidney's whole cadmium content is present in the cortex of the kidney. Lead in the medulla and zinc in the cortex are found in larger quantities. Analyzing the distribution according to sexes it was found that lead in the liver and kidney and cadmium in the kidney and lung of women is significantly lower than in the male. However, the element content in the adrenal is significantly higher for zinc and cadmium in women. The cadmium distribution in the kidney is similar to the essential elements. The correlation of the element pairs is better in the kidney and lung (Zn-Cu is 0.55 in the kidney and 0.51 in the lung).

Cadmium↗

PubMed2002

Uptake of trace elements by rice plants inoculated with Pyricularia oryzae.

The ability of rice plants inoculated with Pyricularia oryzae (P. oryzae) to take up trace elements was studied by the radioactive multitracer technique. Among various elements, only Mn, Co, Zn, Se, Rb, Sr, Tc, and Re were found to be transferred to rice shoots from soil. The concentrations of essential elements, Mn and Zn, in the shoots of rice plants inoculated with P. oryzae were slightly higher than those in the control plant shoots, while Se, Rb, Tc, and Re showed almost the same concentrations for both the shoots.

Ascomycota↗

PubMed2006

Determination of trace elements in herbal tea products and their infusions consumed in Thailand.

Nineteen elements, Mg, Al, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Sr, Sb, Ba, As, Cd, Hg, and Pb, were determined in three types of popular herbal tea products, Gynostemma pentaphyllum, Camellia sinensis, and Morus alba. These herbal tea products, both imported and locally made products, are widely consumed in Thailand and worldwide. Microwave-assisted acid digestion was used for all of the samples, and the element contents were determined by ICP-MS. The concentrations of all elements varied among these herbal teas. Ca and Mg were the most abundant elements in all herbal samples (1384-34070 and 783-7739 mg/kg, respectively). Most elements in these herbal tea powders were also released into the infusions at different percentages depending on types of herbs. G. pentaphyllum infusion contained essential elements (Mg, Ca, V, and Fe) at higher levels than C. sinensis and M.alba infusions. Al and Ni were present at high levels in C. sinensis infusion, and Cd level was high in M. alba infusion. The daily intake of all elements from these herbal tea infusions (three cups/day) is still within the average daily intake. Therefore, it may not produce any health risks for human consumption, if other sources of toxic metal contaminated food are not taken at the same time.

Beverages↗

PubMed1997

The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways.

The 97-kD O-linked glycoprotein, Nup98, is a component of the Xenopus laevis nuclear pore complex and the only vertebrate GLFG nucleoporin identified (Powers, M.A., C. Macauley, F. Masiarz, and D.J. Forbes. 1995. J. Cell Biol. 128:721-736). We have investigated possible roles of xNup98 in the nucleocytoplasmic transport of proteins and RNAs by analyzing the consequences of injecting monospecific polyclonal antibodies to xNup98 into X. laevis oocytes. We show here that nuclear injection of anti-xNup98 inhibited the export of multiple classes of RNAs, including snRNAs, 5S RNA, large ribosomal RNAs, and mRNA. In contrast, the export of tRNA was unaffected. Injection of anti-xNup98 into the oocyte cytoplasm had no effect on export of any of the RNAs. Significantly, nuclear injection of anti-xNup98 antibodies did not inhibit import of either karyophilic proteins or snRNPs. This latter result is in agreement with our previous finding that Nup98 is not an essential element of the protein import pathway. Thus, Nup98 plays a role specifically in RNA export from the nucleus, and it appears to be an essential component of multiple RNA export pathways.

Animals↗

PubMed2002

MAL2, a novel raft protein of the MAL family, is an essential component of the machinery for transcytosis in hepatoma HepG2 cells.

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells.

Actins↗

PubMed1998

An essential secondary structure in the 3' untranslated region of the mouse hepatitis virus genome.

The 3' untranslated regions (3' UTRs) of coronaviruses contain the signals necessary for negative strand RNA synthesis and may also harbor elements essential for positive strand replication and subgenomic RNA transcription. The 3' UTRs of mouse hepatitis virus (MHV) and bovine coronavirus (BCV) are more than 30% divergent. In an effort to learn what parts of these regions might be functionally interchangeable, we attempted to replace the 3' UTR of MHV with its BCV counterpart by targeted RNA recombination. Initially, we tried to substitute the 3' 267 nucleotides (nt) of the 301 nt MHV 3' UTR with the corresponding region of the BCV 3' UTR. This exchange did not yield viable recombinant viruses, and the donor DI RNA was shown to be unable to replicate with MHV as a helper virus. Subsequent analysis revealed that the entire BCV 3' UTR could be inserted into the MHV genome in place of the entire MHV 3' UTR. It resulted that the failure of the initial attempted substitution was due to the inadvertent disruption of an essential conserved bulged stem-loop secondary structure in the MHV and BCV 3' URTs immediately downstream of the N gene stop codon.

3' Untranslated Regions↗

PubMed1990

Deletion-tolerance and trans-splicing of the bacteriophage T4 td intron. Analysis of the P6-L6a region.

Non-directed mutagenesis and phylogenetic comparison suggest that certain elements of the bacteriophage T4 td group Ia intron are dispensable to self-splicing. The L6-P6a-L6a region was identified as a potential non-essential element, and was removed by sequential deletions extending from the L6a loop toward the P6 pairing. Assays for splicing indicate that as long as the P6 pairing is maintained, the 1016 nucleotide td intron can be reduced to less than 250 nucleotides while maintaining function in vivo and in vitro. The P6 pairing appears to be essential for splicing while P6a is not. In addition, a spontaneous pseudorevertant of a splicing-defective deletion was isolated and shown to result from a single nucleotide change in the predicted L6a loop. This genetic suppressor mimics the ability of Mg2+ to reverse the phenotype of the deletion, suggesting that function is restored by structural stabilization of P6. The tolerance of this region to deletion prompted us to split the ribozyme core in L6a, to generate precursors that might function in trans. Indeed, the two half-molecules do associate to form a bimolecular complex that yields accurately ligated exons both in vitro and in vivo. The biological implications of these results, as well as the usefulness of trans-splicing for generating unprocessed precursors in vitro are discussed.

Base Sequence↗