Photoacoustic reconstruction from photothermal measurements including prior information.

Photothermal measurements with an infrared camera enable a fast and contactless part inspection. The main drawback of existing reconstruction methods is the degradation of the spatial resolution with increasing imaging depth, which results in blurred images for deeper lying structures. In this paper, we propose an efficient image reconstruction strategy that allows prior information to be included to overcome the diffusion-based information loss. Following the virtual wave concept, in a first step we reconstruct an acoustic wave field that satisfies the standard wave equation. Therefore, in the second step, stable and efficient reconstruction methods developed for photoacoustic tomography can be used. We compensate for the loss of information in thermal measurements by incorporating the prior information positivity and sparsity. Therefore, we combine circular projections with an iterative regularization scheme. Using simulated and experimental data, this work demonstrates that the quality of the reconstruction from photothermal measurements can be significantly enhanced.

Reduced proliferation of CD34(+) cells from patients with acute myeloid leukemia after gene transfer of INPP5D.

Acute myeloid leukemia (AML) is a malignant disease characterized by deregulated proliferation of immature myeloid cells. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently detected in approximately 50-70% of AML patients. The gene INPP5D encodes the SH2-containing inositol 5-phosphatase 1 (SHIP1), which is a negative regulator of PI3K/AKT signaling. After lentiviral-mediated gene transfer of INPP5D into CD34(+) cells derived from AML patients (n=12) the granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent proliferation was reduced in all samples analyzed (average 86%; range 72-93%). An enzymatically inactive form of SHIP1 (D672A) had no effect. In addition, SHIP1 reduced the autonomous proliferation of CD34(+) cells from a patient with a secondary AML who had a very high peripheral blast count (300 x 10(9) l(-1)). These data show that SHIP1 can effectively block GM-CSF-dependent and autonomous proliferation of AML cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP(+)) is an essential regulator of T-lymphocyte Ca(2+)-signaling.

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP(+)) dose-dependently stimulated intracellular Ca(2+)-signaling. At a concentration of 10 nM NAADP(+) evoked repetitive and long-lasting Ca(2+)-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca(2+)-peak followed by trains of smaller Ca(2+)-oscillations was observed. Higher concentrations of NAADP(+) (1 and 10 microM) gradually reduced the initial Ca(2+)-peak, and a complete self-inactivation of Ca(2+)-signals was seen at 100 microM. The effect of NAADP(+) was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4, 5-trisphosphate- and cyclic adenosine diphosphoribose-mediated Ca(2+)-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP(+). Most importantly, microinjection of a self-inactivating concentration of NAADP(+) completely abolished subsequent stimulation of Ca(2+)-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP(+) Ca(2+)-release system is essential for T-lymphocyte Ca(2+)-signaling.

Inhibition of phosphatases and increased Ca2+ channel activity by inositol hexakisphosphate.

Inositol hexakisphosphate (InsP6), the dominant inositol phosphate in insulin-secreting pancreatic beta cells, inhibited the serine-threonine protein phosphatases type 1, type 2A, and type 3 in a concentration-dependent manner. The activity of voltage-gated L-type calcium channels is increased in cells treated with inhibitors of serine-threonine protein phosphatases. Thus, the increased calcium channel activity obtained in the presence of InsP6 might result from the inhibition of phosphatase activity. Glucose elicited a transient increase in InsP6 concentration, which indicates that this inositol polyphosphate may modulate calcium influx over the plasma membrane and serve as a signal in the pancreatic beta cell stimulus-secretion coupling.

Regulation of Ca2+ influx in myeloid cells. Role of plasma membrane potential, inositol phosphates, cytosolic free [Ca2+], and filling state of intracellular Ca2+ stores.

To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by microperfusion through the patch pipette. In voltage-clamped cells, [Ca2+]i elevations with a sustained phase could be induced by (a) the chemoattractant receptor agonist FMLP, (b) the Ca(2+)-releasing second messenger myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3], as well as its nonmetabolizable analogues, and (c) the Ca(2+)-ATPase inhibitor cyclopiazonic acid, which depletes intracellular Ca2+ stores. In the absence of extracellular Ca2+, responses to all stimuli were short-lasting, monophasic transients; however, subsequent addition of Ca2+ to the extracellular medium led to an immediate [Ca2+]i increase. In all cases, the sustained phase of the [Ca2+]i elevations could be inhibited by millimolar concentrations of extracellular Ni2+, and its amplitude could be decreased by depolarization of the plasma membrane. Thus, the sustained phase of the Ca2+ elevations was due to Ca2+ influx through a pathway sensitive to the electrical driving force and to Ni2+. No Ca2+ influx could be observed after (a) plasma membrane depolarization in resting cells, (b) an imposed [Ca2+]i transient independent of receptor activation, or (c) microperfusion of myo-inositol(1,3,4,5)tetrahisphosphate (Ins(1,3,4,5)P4). Also, Ins(1,3,4,5)P4 did not have additive effects when co-perfused with a submaximal concentration of Ins(1,4,5)P3. Our results suggest that, in myeloid cells, activation of chemoattractant receptors induces an electrogenic, Ni(2+)-sensitive Ca2+ influx via generation of Ins(1,4,5)P3. Ins(1,4,5)P3 might activate Ca2+ influx directly, or by depletion of intracellular Ca2+ stores, but not via [Ca2+]i increase or Ins(1,3,4,5)P4 generation.

p53 domains: structure, oligomerization, and transformation.

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

A novel intron element operates posttranscriptionally To regulate human N-myc expression.

Precisely regulated expression of oncogenes and tumor suppressor genes is essential for normal development, and deregulated expression can lead to cancer. The human N-myc gene normally is expressed in only a subset of fetal epithelial tissues, and its expression is extinguished in all adult tissues except transiently in pre-B lymphocytes. The N-myc gene is overexpressed due to genomic amplification in the childhood tumor neuroblastoma. In previous work to investigate mechanisms of regulation of human N-myc gene expression, we observed that N-myc promoter-chloramphemicol acelyltransferase reporter constructs containing sequences 5' to exon 1 were active in all cell types examined, regardless of whether endogenous N-myc RNA was detected. In contrast, inclusion of the first exon and a portion of the first intron allowed expression only in those cell types with detectable endogenous N-myc transcripts. We investigated further the mechanisms by which this tissue-specific control of N-myc expression is achieved. Using nuclear run-on analyses, we determined that the N-myc gene is actively transcribed in all cell types examined, indicating a posttranscriptional mode of regulation. Using a series of N-myc intron 1 deletion constructs, we localized a 116-bp element (tissue-specific element [TSE]) within the first intron that directs tissue-specific N-myc expression. The TSE can function independently to regulate expression of a heterologous promoter-reporter minigene in a cell-specific pattern that mirrors the expression pattern of the endogenous N-myc gene. Surprisingly, the TSE can function in both sense and antisense orientations to regulate gene expression. Our data indicate that the human N-myc TSE functions through a posttranscriptional mechanism to regulate N-myc expression.

Nematodes in wastewater biofilms--appearance and density of species in three biofilter reactors.

Population dynamics of nematode species in biofilms of three different biofilter reactors, differing in size (pilot/laboratory scale), operation mode and biofilm carrier, were studied over a period of 1 year. In the biofilm suspension of the pilot system mean nematode density was 118individuals/ml and average biomass 15microg wet weight/ml. Higher mean abundance was found in the two laboratory systems with 2380 and 4411individuals/ml. Mean biomass in the laboratory systems ranged from 209 to 330microg wet weight/ml. There were marked temporal differences in appearance and density of nematode species in all three biofilters. Number of species observed was 3 in the laboratory systems and 5 in the pilot system. The fastest growing species (Paroigolaimella bernensis and Diplogasteritus nudicapitatus) were observed in the pilot reactor in contrast to the more slowly growing species (Diploscapter coronatus and Acrostichus sp.), which dominated in the laboratory reactors. Sexual reproduction was found for all species but of Diploscapter coronatus. When comparing life history traits of the different species with the environmental conditions in the reactors, it seems that the unstable conditions in the pilot reactor favor the fast growing species whereas the stable environment in the laboratory systems allows the growth of species with longer generation times.

Serine phosphorylation in the NH2 terminus of p53 facilitates transactivation.

Murine tumor suppressor p53 is phosphorylated in the NH2-terminal transactivating domain at serines 9, 18, and 37. Change of any one of these serines to either alanine or aspartic acid did not alter p53 suppression of transformation of rat embryo fibroblasts by activated ras and E1A. Change of any two of these serines to alanines, however, led to a significant decrease in suppressor function. Substitution of alanines for all three serines caused the most severe loss of suppression and also reduced transactivation functions. The triple substitution had no apparent effects on intracellular accumulation or localization of p53, oligomerization, DNA binding, or interaction with the TFIID TATA-binding protein. In contrast, triple substitution of aspartic acid for serines 9, 18, and 37 had minimal effects on suppression and transactivation by p53. These results argue strongly that phosphorylation of serines 9, 18, and 37 facilitates the suppression and transactivation functions of p53.

Functional expression and characterisation of a new human phosphatidylinositol 4-kinase PI4K230.

By constructing DNA probes we have identified and cloned a human PtdIns 4-kinase, PI4K230, corresponding to a mRNA of 7.0 kb. The cDNA encodes a protein of 2044 amino acids. The C-terminal part of ca. 260 amino acids represents the catalytic domain which is highly conserved in all recently cloned PtdIns 4-kinases. N-terminal motifs indicate multiple heterologous protein interactions. Human PtdIns 4-kinase PI4K230 expressed in vitro exhibits a specific activity of 58 micromol mg-1min-1. The enzyme expressed in Sf9 cells is essentially not inhibited by adenosine, it shows a high Km for ATP of about 300 microM and it is half-maximally inactivated by approximately 200 nM wortmannin. These data classify this enzyme as type 3 PtdIns 4-kinase. Antibodies raised against the N-terminal part moderately activate and those raised against the C-terminal catalytic domain inhibit the enzymatic activity. The coexistence of two different type 3 PtdIns 4-kinases, PI4K92 and PI4K230, in several human tissues, including brain, suggests that these enzymes are involved in distinct basic cellular functions.

Beta1-integrins mediate Ca2+-signalling and T cell spreading via divergent pathways.

Interaction of Jurkat T-lymphocytes with two extracellular matrix (ECM) proteins of the basement membrane, laminin or collagen type IV, combined with poly-L-lysine resulted in a strong adhesion, a highly increased intracellular Ca2+-concentration ([Ca2]i), as compared to cells on laminin or collagen type IV alone and in spreading of the cells. The strong adhesion was independent of an increase in [Ca2+]i, was not mediated by a beta1-integrin, and was due to charge interaction between the positively charged polyaminoacid and the negatively charged cell surface. The latter was confirmed by substitution of poly-L-lysine by other positively charged polyaminoacids. In contrast, Ca+-signalling and spreading of the cells adhering to laminin or collagen type IV combined with poly-L-lysine was completely blocked by anti-beta1 mAb. However, spreading of the cells was independent of an increase in [Ca2+]i suggesting divergent signal transduction pathways leading to Ca2+-signalling and spreading of the cells. We elucidated these signal transduction pathways by inhibition of key enzymes involved. The tyrosine kinase inhibitor genistein blocked Ca2+-signalling as well as spreading, whereas inhibitors of PKC (calphostin C, GF109203x), PLCgamma (U73122) and PLA2 (bromophenacyl-bromide (BPB), 3-[4-octadecyl)benzoyl]acrylic acid (OBAA)) selectively blocked spreading of the cells.

Mechanisms involved in alpha6beta1-integrin-mediated Ca(2+) signalling.

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting alpha6beta1-integrin-mediated Ca(2+) signalling. Both Ca(2+) release from thapsigargin-sensitive Ca(2+) stores and capacitative Ca(2+) entry via Ca(2+) channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of alpha6beta1-integrin-mediated Ca(2+) signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca(2+)-releasing second messengers D-myo-inositol 1,4,5-trisphosphate (InsP3) and cADPR.

Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

Review of the 2012 Epizootic Hemorrhagic Disease Outbreak in Domestic Ruminants in the United States.

An unusually large number of cases of Epizootic hemorrhagic disease (EHD) were observed in United States cattle and white-tailed deer in the summer and fall of 2012. USDA APHIS Veterinary Services area offices were asked to report on foreign animal disease investigations and state diagnostic laboratory submissions which resulted in a diagnosis of EHD based on positive PCR results. EHD was reported in the following species: cattle (129 herds), captive white-tailed deer (65 herds), bison (8 herds), yak (6 herds), elk (1 herd), and sheep (1 flock). A majority of the cases in cattle and bison were found in Nebraska, South Dakota, and Iowa. The majority of cases in captive white-tailed deer were found in Ohio, Iowa, Michigan, and Missouri. The most common clinical sign observed in the cattle and bison herds was oral lesions. The major observation in captive white-tailed deer herds was death. Average within-herd morbidity was 7% in cattle and bison herds, and 46% in captive white-tailed deer herds. The average within-herd mortality in captive white-tailed deer herds was 42%.

Unique properties of the capacitative Ca(2+)-entry antagonist LU 52396: its inhibitory activity depends on the activation state of the cells.

The pharmacological properties of the recently described antagonist for capacitative Ca2+ entry LU 52396 were investigated and compared to known Ca2+ antagonists in Jurkat T-lymphocytes. In the first set of experiments, cells were stimulated with the anti-CD3 monoclonal antibody OKT3 and, subsequently, Ca2+ antagonists were added. Under such conditions SK-F 96365, econazole, nitrendipine and ZnCl2 dose-dependently antagonized Ca2+ signaling, whereas LU 52396 in concentrations up to 100 microM did not. In contrast, when LU 52396 was added a few minutes before OKT3, a dose-dependent inhibition of the OKT3-stimulated Ca2+ signals by LU 52396 was observed. Likewise, by prior addition of LU 52396 to thapsigargin-stimulated Jurkat T cells, a dose-dependent inhibition of Ca2+ signals was achieved. The IC50 value of LU 52396 for both agonists was about 5 microM. LU 52396 also inhibited Jurkat T cell proliferation, but showed cytotoxic effects at concentrations > 50 microM. Our data indicate that, in contrast to the other Ca2+ antagonists SK-F 96365, econazole, nitrendipine and ZnCl2, LU 52396 recognized the channel for capacitative Ca2+ entry only when intracellular Ca2+ was low and the channel was in its closed state.

Suppression of thyrotropin receptor-G protein-phospholipase C coupling by activation of protein kinase C in thyroid carcinoma cells.

In human thyroid follicular cells TSH exerts its action on growth and function at least via two distinct pathways, the adenylate cyclase cascade and the phospholipase Cbeta (PLCbeta)-mediated inositol phosphate generation. We investigated the effect of TSH on activation of phosphoinositide hydrolysis and inositol phosphate generation by PLCbeta in HTh74 thyroid carcinoma cells that express functional TSH receptors and in HTC-TSHr thyroid carcinoma cells that are devoid of endogenous TSH receptors but express recombinant human TSH receptors. In both cell lines, TSH up to concentrations of 300 mU/ml failed to stimulate myo-inositol 1,4,5-trisphosphate and myo-inositol-tetrakisphosphate generation, but led to a decrease in these compounds within 1 min of stimulation. However, ATP and bradykinin increased concentrations of inositol phosphates in both thyroid carcinoma cell lines. In contrast, in differentiated FRTL5 thyroid cell line and CHO-TSHr cell line expressing recombinant human TSH receptors, TSH elicited a significant increase in myo-inositol 1,4,5-trisphosphate and its metabolic derivatives. However, when HTC-TSHr cells were pretreated with calphostin C or staurosporine, inhibitors of protein kinase C, a TSH concentration of 20 mU/ml enhanced generation of inositol phosphates in these cells. From our data we conclude that in HTC-TSHr and HTh74 thyroid carcinoma cells, the coupling within the TSH receptor-Gq protein-PLCbeta signaling pathway is impaired compared to that in nontransformed cells. It is conceivable that this is at least in part dependent on the level of protein kinase C activation in these cells.

A novel A-isoform-like inositol 1,4,5-trisphosphate 3-kinase from chicken erythrocytes exhibits alternative splicing and conservation of intron positions between vertebrates and invertebrates.

Based on the partial peptide sequence of inositol 1,4, 5-trisphosphate 3-kinase purified with 135 000-fold enrichment from chicken erythrocytes, cDNA-fragments were cloned by RT-PCR using degenerate oligonucleotides. Subsequent hybridization screening of an embryonic chicken cDNA library and 5'-RACE yielded a cDNA-contig of 2418 bp, encoding a 452 amino acid protein. The amino acid sequence shows the highest degree of homology with A-isoforms of inositol 1,4,5-trisphosphate 3-kinase (65% identities), whereas homology towards B and C isoforms was lower (57% and 52% amino acid identities respectively). These findings reveal a new tissue-specific pattern of A-isoform expression, a form which so far has only been found in brain and testes. Two overlapping lambda-genomic clones for chicken inositol 1,4,5-trisphosphate 3-kinase, isolated by hybridization screening, covered 18 499 bp of genomic sequence. This contig included four exons: three of them were present in all cDNA clones, whereas one was only represented in a single cDNA clone. In addition, the sequence of the latter differed from the other cDNAs by an in-frame deletion of 72 bp within the coding region for the catalytic domain of the enzyme. This divergent cDNA suggests the existence of alternative splice products, at least in embryonic tissue.A comparison of the position of introns, with the respective introns known from the corresponding gene from Caenorhabditis elegans, revealed a high degree of conservation of intron positions between vertebrates and invertebrates. Functional data for the enzyme suggests that the conserved exons represent defined functional protein modules.

The only inositol tetrakisphosphate detectable in avian erythrocytes is the isomer lacking phosphate at position 3: a NMR study.

Avian red blood cells contain a millimolar amount of inositol polyphosphate which plays a role as an allosteric effector of hemoglobin. We confirmed the structure of this substance by NMR techniques as purely myo-inositol 1,3,4,5,6-pentakisphosphate. Based on present knowledge this effector is synthesized from inositol trisphosphate by successive phosphorylation. In a search for biosynthetic and degradative intermediates of inositol pentakisphosphate we found only one inositol tetrakisphosphate isomer. Its structure was unambiguously assigned by proton, 13C- and 31P-NMR to myo-inositol 1,4,5,6-tetrakisphosphate. As in mammalian cells the major inositol tetrakisphosphate isomer is myo-inositol 1,3,4,5-tetrakisphosphate, there seem to be differences between avian red blood cells and mammalian cells in the routes of inositol polyphosphate formation and/or degradation.

Phosphofructokinase is a calmodulin binding protein.

A trial to purify myosin light chain kinase from crude myosin led to the isolation of a Mr 85 000 calmodulin binding protein different from this enzyme. Because it showed inherent phosphofructokinase activity we investigated its relation to this enzyme. We demonstrated identity to phosphofructokinase by a close to identical amino acid composition, by antigenic identity and a set of completely identical peptide maps. The calmodulin binding property was also shown for a fraction of the enzyme prepared by standard methods. First experiments show that Ca2+--calmodulin is a potent regulator of phosphofructokinase polymerization.

Skeletal muscle myosin light chain kinase. A refined structural model.

A hydrodynamic, enzymatic and CD spectroscopic study of skeletal muscle myosin light chain kinase, three proteolytic fragments and corresponding complexes with calmodulin was performed. A refined shape model was built for the enzyme. It was shown that a head-and-tail structure is formed from two major fragments which are aligned end-to-end. The one fragment (Mr 36000) is compact, of high alpha-helix content and contains the catalytic center with the light chain and the calmodulin binding domains. The other fragment (Mr 33000) with unknown function is asymmetric (a/b greater than 10), of low alpha-helix and of unusually high proline content.