Predicting cadmium fractions in agricultural soils using proximal sensing techniques.

Cadmium (Cd) accumulation in agricultural systems has caused global environmental and health concerns. Application of phosphate fertiliser to sustain plant production unintentionally accumulated Cd in agricultural soils over time. Rapid and cost-effective Cd monitoring in these soils will help to inform Cd management practices. Compared to total Cd analysis, examining chemical fractions by sequential extraction methods can provide information on the origin, availability, and mobility of soil Cd, and to assess the potential plant Cd uptake. A total of 87 air-dried topsoil (0-15 cm) samples from pastoral farms with a history of long-term application of phosphate fertiliser were analysed using wet chemistry methods for total Cd and Cd forms in exchangeable, acid soluble, metal oxides bound, organic matter bound, and residual fractions. The data acquired using three proximal sensing techniques, visible-near-infrared (vis-NIR), mid-infrared (MIR), and portable X-ray fluorescence (pXRF) spectroscopy were used as input for partial least squares regression to develop models predicting total Cd and Cd fractions. The average total Cd concentration was 0.58 mg Cd/kg soil. For total Cd, cross-validation (cv) results of models using individual vis-NIR, MIR, and pXRF data performed with normalised root mean squared error (nRMSEcv) of 26%, 30%, and 31% and concordance correlation coefficient (CCCcv) of 0.85, 0.77, and 0.75, respectively. For exchangeable Cd, model using MIR data performed with nRMSEcv of 40% and CCCcv of 0.57. For acid soluble and organic matter bound Cd, models using vis-NIR data performed with nRMSEcv of 11% and 33% and CCCcv of 0.97 and 0.84, respectively. Reflectance spectroscopy techniques could potentially be applied as complementary tools to estimate total Cd and plant available and potentially available Cd fractions for effective implementation of Cd monitoring programmes.

Surveillance and movements of Virginia opossum (Didelphis virginiana) in the bovine tuberculosis region of Michigan.

Wildlife reservoir hosts of bovine tuberculosis (bTB) include Eurasian badgers (Meles meles) and brushtail possum (Trichosurus vulpecula) in the UK and New Zealand, respectively. Similar species warrant further investigation in the northern lower peninsula of Michigan, USA due to the continued presence of bTB on cattle farms. Most research in Michigan, USA has focused on interactions between white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus) for the transmission of the infectious agent of bTB, Mycobacterium bovis, due to high deer densities and feeding practices. However, limited data are available on medium-sized mammals such as Virginia opossum (Didelphis virginiana; hereafter referred to as opossum) and their movements and home range in Michigan near cattle farms. We conducted surveillance of medium-sized mammals on previously depopulated cattle farms for presence of M. bovis infections and equipped opossum with Global Positioning System (GPS) technology to assess potential differences in home range between farms inside and outside the bTB core area that has had cattle test positive for M. bovis. On farms inside the bTB core area, prevalence in opossum was comparable [6%, 95% confidence interval (CI) 2.0-11.0] to prevalence in raccoon (Procyon lotor; 4%, 95% CI 1.0-9.0, P=0.439) whereas only a single opossum tested positive for M. bovis on farms outside the bTB core area. The prevalence in opossum occupying farms that had cattle test positive for M. bovis was higher (6.4%) than for opossum occupying farms that never had cattle test positive for M. bovis (0.9%, P=0.01). Mean size of home range for 50% and 95% estimates were similar by sex (P=0.791) both inside or outside the bTB core area (P=0.218). Although surveillance efforts and home range were not assessed on the same farms, opossum use of farms near structures was apparent as was selection for farms over surrounding forested habitats. The use of farms, stored feed, and structures by opossum, their ability to serve as vectors of M. bovis, and their propensity to ingest contaminated sources of M. bovis requires additional research in Michigan, USA.

'Generic visuals' of Covid-19 in the news: Invoking banal belonging through symbolic reiteration.

In the early days of the Covid-19 pandemic, images of the virus molecule and 'flatten-the-curve' line charts were inescapable. There is now a vast visual repertoire of vaccines, people wearing face masks in everyday settings, choropleth maps and both bar and line charts. These 'generic visuals' circulate widely in the news media and, however unremarkable, play an important role in representing the crisis in particular ways. We argue that these generic visuals promote banal nationalism, localism and cosmopolitanism in the face of the crisis, and that they do so through the symbolic reiteration of a range of visual resources across news stories. Through an analysis of three major news outlets in the UK, we examine how generic visuals of Covid-19 contribute to these banal visions and versions of belonging and, in doing so, also to foregrounding the role of the state in responding to the crisis.

Immunoassay by differential pulse polarography.

An immunological method based on labeling an antigen with an electroactive group detectable by differential pulse polarography has been demonstrated. Estriol labeled with mercuric acetate is electroactive, giving a reduction wave at -300 millivolts versus a standard calomel electrode. Addition of estriol antibody to 4-mercuric acetate estriol diminishes the peak current as a result of the antigen-antibody binding reaction. Separation of free-labeled estriol from antibody-bound-labeled antigen is unnecessary. The method is potentially useful as an analytical immunological technique.

Enhanced phosphorylation of p53 by ATM in response to DNA damage.

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase-related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

DNA damage and the DNA-activated protein kinase.

DNA-activated protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that is activated in vitro by DNA fragments. The cellular targets of DNA-PK are nuclear, DNA-binding, regulatory proteins including Sp1, Fos, Jun, Myc, the tumor suppressor protein p53, and RNA polymerase II. These characteristics suggest a role for DNA-PK in coordinating nuclear processes and as a modulator of checkpoint mechanisms activated by DNA damage.

Can ACG serve as an initiation codon for protein synthesis in eucaryotic cells?

An ACG codon, which replaces the AUG codon used to initiate the synthesis of bacteriophage T7 gene 0.3 protein, was shown to function as a low-efficiency initiation codon in a wheat germ cell-free protein-synthesizing system.

Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.

Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53.

Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.

Suramin affects DNA synthesis in HeLa cells by inhibition of DNA polymerases.

Suramin, a polysulfonated naphthylurea widely used in the treatment of trypanosomiasis and onchocerciasis, is currently being investigated as an antitumor agent for the treatment of advanced cancer. Suramin exerts a wide variety of biological effects. We have shown that suramin inhibits cell proliferation and DNA synthesis in cultured HeLa cells. The replication in vitro of SV40 DNA is completely abolished by 40 microM suramin. The inhibition of DNA replication is due to inhibition of DNA polymerases alpha and delta, the replicative enzymes in eukaryotic cells. DNA polymerase alpha is sensitive to lower concentrations of suramin [concentration to achieve 50% inhibition (IC50) of 8 microM] than is DNA polymerase delta (IC50 36 microM), whereas DNA polymerase beta is relatively insensitive to the drug (IC50 of 90 microM). Suramin inhibits other replicative DNA polymerases such as Escherichia coli polymerase I (Klenow fragment) and Thermus aquaticus polymerase. Suramin is noncompetitive with both substrate deoxyribonucleotides and template-primers with respect to DNA polymerase inhibition. Much lower concentrations (8-30 microM) of the drug are required for 50% inhibition of DNA polymerases than for 50% inhibition of other enzymes such as protein kinase C and reverse transcriptase. These results show an important biological effect of this drug and indicate the need for more studies before its clinical use as an antitumor agent.

RNA is covalently linked to SV40 large T antigen.

RNA is shown to be covalently linked to the large tumor antigen (TAg) of simian virus 40 (SV40). Proteolytic digestion of TAg, isolated in the presence of ribonuclease inhibitors from SV40 transformed Balb/c mouse cells, generated a specific phosphopeptide of high charge heterogeneity that was strongly retained on DEAE-cellulose in the presence of 7 M urea. Hydrolysis of this peptide with RNAase released the four standard ribonucleotide monophosphates. Analysis of peptide digestion products showed that the RNA is attached to TAg through a phosphodiester linkage between the beta-hydroxyl of a serine and the 5' phosphate of an invariant cytidine residue. The methods applied to SV40 TAg can be applied to other proteins, including cellular oncogene products, to investigate the possibility of covalent protein-RNA interactions.

Differential stability of the DNA-activated protein kinase catalytic subunit mRNA in human glioma cells.

DNA-dependent protein kinase (DNA-PK) functions in double-strand break repair and immunoglobulin [V(D)J] recombination. We previously established a radiation-sensitive human cell line, M059J, derived from a malignant glioma, which lacks the catalytic subunit (DNA-PKcs) of the DNA-PK multiprotein complex. Although previous Northern blot analysis failed to detect the DNA-PKcs transcript in these cells, we show here through quantitative studies that the transcript is present, albeit at greatly reduced (approximately 20x) levels. Sequencing revealed no genetic alteration in either the promoter region, the kinase domain, or the 3' untranslated region of the DNA-PKcs gene to account for the reduced transcript levels. Nuclear run-on transcription assays indicated that the rate of DNA-PKcs transcription in M059J and DNA-PKcs proficient cell lines was similar, but the stability of the DNA-PKcs message in the M059J cell line was drastically (approximately 20x) reduced. Furthermore, M059J cells lack an alternately spliced DNA-PKcs transcript that accounts for a minor (5-20%) proportion of the DNA-PKcs message in all other cell lines tested. Thus, alterations in DNA-PKcs mRNA stability and/or the lack of the alternate mRNA may result in the loss of DNA-PKcs activity. This finding has important implications as DNA-PKcs activity is essential to cells repairing damage induced by radiation or radiomimetric agents.

DNA-PK, the DNA-activated protein kinase, is differentially expressed in normal and malignant human tissues.

DNA-PK is a nuclear, serine/threonine protein kinase required for repairing DNA double-strand breaks and for V(D)J recombination. To determine the distribution of DNA-PK in human tissues, we assayed paraffin-embedded sections of normal and cancerous tissues for DNA-PKcs and Ku80 by immunohistochemistry. We also assayed for Brca2, a human tumor suppressor gene that is implicated in the repair of DNA strand-breaks. Brca2 was strongly expressed in epithelial cells of the breast, endometrium, and thymus, in tingible body macrophages of follicular germinal centers of lymphoid tissue, and in reticuloendothelial cells in the spleen. DNA-PKcs and Ku80 expression was usually parallel, but both were expressed in a highly cell- and tissue-specific manner. The highest levels were observed in spermatogenic cells (but not in spermatozoa), and in neurons and glial cells of the central and autonomic nervous system. Neither protein was consistently expressed in liver nor in resting mammary epithelium, but lactating breast epithelium was strongly positive for DNA-PKcs and Ku80. In contrast to established human cell cultures, expression between cells in the same tissue was highly selective in the epidermis, exocrine pancreas, renal glomeruli, the red pulp of the spleen, and within cellular compartments of tonsils, lymph nodes, and thymus. Most cancerous tissues were consistently positive for DNA-PKcs and Ku80, except invasive carcinoma of the breast. DNA-PKcs, Ku80, and Ku70 mRNAs were expressed in all normal tissues with relatively little variation in levels. Our results suggest that the apparent absence of DNA-PKcs and Ku80 from some cells or tissues is a consequence of post-transcriptional mechanisms that regulate protein levels.

Mutation of the serine 15 phosphorylation site of human p53 reduces the ability of p53 to inhibit cell cycle progression.

Overexpression of wild-type p53 prevents cells from entering the S phase of the cell cycle. The amino-terminal transactivation region of p53 is phosphorylated by several protein kinases, including DNA-PK, a nuclear serine/threonine protein kinase that in vitro requires DNA for activity. DNA-PK was recently shown to phosphorylate serines 15 and 37 of human p53 (Lees-Miller et al., 1992. Mol. Cell. Biol., 12, 5041-5049). To prevent phosphorylation at these sites, mutants were constructed that changed the codons for serine 15 or serine 37 to alanine codons. Expression of p53-Ala-37 in stably transformed T98G cells blocked progression of the cells into S phase as well as did the expression of wild-type p53. In contrast, p53-Ala-15 was partially defective in blocking cell cycle progression. Several cell clones transformed with the mutant p53-Ala-15 gene expressed normal levels of p53 mRNA but accumulated little or no detectable p53 protein. However, by using a transient expression system driven by a strong cytomegalovirus promoter, we showed that the inability of p53-Ala-15 to fully block cell cycle progression was not due to inadequate levels of expression or to a failure of the mutant protein to accumulate in the nucleus. These results suggest that phosphorylation of Ser-15 may affect p53 function.

The carboxy-terminal serine 392 phosphorylation site of human p53 is not required for wild-type activities.

Wild-type p53 functions in the G1 DNA damage checkpoint pathway by activating gene transcription and preventing cell cycle progression. Others reported that mutation of the serine 386 codon in mouse p53 abolished its ability to suppress growth. Serine 386 of murine p53 and the homologous residue of human p53, serine 392, are phosphorylated in vivo and can be phosphorylated in vitro by casein kinase II (CKII). We constructed mutants that changed serine 392 of human p53 to alanine (p53-S392A) or aspartic acid (p53-S392D); cotransfection of both these mutants with a reporter gene carrying a p53-responsive element into the p53-null Saos-2 cell line activated transcription as well as did wild-type p53. Furthermore, both mutants blocked cell cycle progression after transient transfection in these cells. A stable derivative of the T98G human glioblastoma cell line was established that expressed p53-S392A in response to dexamethasone. Overexpression of this mutant activated transcription of the endogenous waf1 (also called cip1) and mdm2 genes to the same extent as wild-type p53 and also produced growth arrest. Finally, p53-S392A and p53-S392D suppressed foci formation by activated ras and adenovirus E1A oncogenes as efficiently as did wild-type p53. Thus, unlike mutants that altered the serine 15 phosphorylation site, elimination of the serine 392 phosphorylation site had no discernible effect on p53 function. We conclude that neither phosphorylation nor RNA attachment to serine 392 are required for human p53's ability to suppress cell growth or to activate transcription in vivo.

Adenovirus interaction with its cellular receptor CAR.

Representative adenoviruses from four of the five major virus subgroups have been shown to interact with the 46-kDa coxsackievirus and adenovirus receptor (CAR) that is widely expressed on many human cell types, suggesting that the ability to bind CAR may be a conserved feature of many of the approximately 50 known adenovirus serotypes. Receptor binding is a function of the distal 'knob' domain of the trimeric viral fiber protein. Here we review recent structural characterizations of knob, CAR and knob-CAR complexes, and we discuss how knob architecture may have evolved to accommodate opposing selective pressures to vary antigenic structure while conserving receptor binding specificity. In contrast to the hypervariability of the solvent-exposed surface of knob, the CAR receptor was found to be non-polymorphic.

Crystallization and preliminary X-ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor.

Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor. The crystals (space group P3(1)21 or P3(2)21, one molecule per asymmetric unit, a = b = 41.3 angstrum, c = 197.0 angstrum) grew in solutions containing 20-40% 2-methyl-2,4-pentanediol (MPD), 0.1-0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0-7.5. Diffraction data (84% complete to 2.2 angstrum resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 x 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 angstrum resolution with Rmerge values ranging 0.05-0.07, have been collected at three X-ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.

The nuclear serine/threonine protein kinase DNA-PK.

The DNA-activated protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that phosphorylates DNA-binding proteins, including several transcription factors. DNA-PK is one of a very few enzymes known to be regulated through interaction with DNA that does not have DNA as a template or substrate. We suggest that DNA-PK may function in cell homeostasis, in part through the modulation of transcriptional activity.

Alternate splice-site utilization in the gene for the catalytic subunit of the DNA-activated protein kinase, DNA-PKcs.

Analysis of polyA-selected RNAs and cDNA clones from several human cell lines revealed the presence of a 93-bp exon in the PI kinase homology region of DNA-PKcs that was not present in the cDNA sequence derived from HeLa cell cDNA clones. RT-PCR showed that most DNA-PKcs mRNAs in human cells have this exon. Thus, the nascent DNA-PKcs polypeptide is composed of 4127 aa and has a predicted mol. wt. of 469021 (470 kDa).

Ozone dose mapping and the utility of models.

In Europe the monitoring of ozone doses to growing crops is based on measurement of AOT40, the annual accumulated excess ozone concentration over a threshold of 40 ppb, aggregated over the growing season. To show the extent of ozone pollution it is desirable to construct maps of AOT40. However, data are limited and there is large inter-annual variation, so what is to be mapped is very variable, and our knowledge of it is limited. This paper describes a spatially referenced random effects model which appears able to describe many features of the data and our uncertainty about them. The problem of translating this or similar models into a map faithfully representing our knowledge is considered, as are some questions it raises about decision-makers' and the public's need for and use of technical models.