Ultrasonic testing of thick and thin Inconel 625 alloys manufactured by laser powder bed fusion.

Additive manufacturing of alloys enables low-volume production of functional metallic components with complex geometries. Ultrasonic testing can ensure the quality of these components and detect typical defects generated during laser powder bed fusion (LPBF). However, it is difficult to find a single ultrasonic inspection technique that can detect defects in the large variety of geometries generated using LPBF. In this work, phased array ultrasonic testing (PAUT) is suggested to inspect thick LPBF components, while guided waves are explored for thin curved ones. PAUT is used to detect cylindrical lack of fusion defects in thick LPBF rectangular parts. Practical defects are generated by reducing the laser power at prespecified locations in the samples. The defects' shape and density are verified using optical microscopy and X-ray computed tomography. Partially fused defects down to 0.25 mm in diameter are experimentally detected using a 10 MHz PAUT probe with the total focusing method post-processing. The experimental results are compared to defect images predicted by finite element simulations. For thin components with curved geometry, guided waves are used to detect powder-filled cylindrical defects. The waves are generated using piezoelectric transducers, and the spatiotemporal wavefield is measured using a scanning laser Doppler vibrometer. Using root-mean-square imaging of the wavefield, defects down to 1 mm are clearly detected despite the complex internal features in the samples.

Vibration-based elastic parameter identification of the diploë and cortical tables in dry cranial bones.

Various human skull models feature a layered cranial structure composed of homogeneous cortical tables and the inner diploë. However, there is a lack of fundamental validation work of such three-layer cranial bone models by combining high-fidelity computational modeling and rigorous experiments. Here, non-contact vibration experiments are conducted on an assortment of dry bone segments from the largest cranial bone regions (parietal, frontal, occipital, and temporal) to estimate the first handful of modal frequencies and damping ratios, as well as mode shapes, in the audio frequency regime. Numerical models that consider the cortical tables and the diploë as domains with separate isotropic material properties are constructed for each bone segment using a routine that identifies the cortical table-diploë boundaries from micro-computed tomography scan images, and reconstructs a three-dimensional geometry layer by layer. The material properties for cortical tables and diploë are obtained using a Hounsfield Unit-based mass density calculation combined with a parameter identification scheme for Young's modulus estimation. With the identified parameters, the average error between experimental and numerical modal frequencies is 1.3% and the modal assurance criterion values for most modes are above 0.90, indicating that the layered model is suitable for predicting the vibrational behavior of cranial bone. The proposed layered modeling and identified elastic parameters are also useful to support computational modeling of cranial guided waves and mode conversion in medical ultrasound. Additionally, the diploë elastic properties are rarely reported in the literature, making this work a fundamental characterization effort that can guide in the selection of material properties for human head models that consider layered cranial bone.

Coherent virtual absorption of elastodynamic waves.

Absorbers suppress reflection and scattering of an incident wave by dissipating its energy into heat. As material absorption goes to zero, the energy impinging on an object is necessarily transmitted or scattered away. Specific forms of temporal modulation of the impinging signal can suppress wave scattering and transmission in the transient regime, mimicking the response of a perfect absorber without relying on material loss. This virtual absorption can store energy with large efficiency in a lossless material and then release it on demand. Here, we extend this concept to elastodynamics and experimentally show that longitudinal motion can be perfectly absorbed using a lossless elastic cavity. This energy is then released symmetrically or asymmetrically by controlling the relative phase of the impinging signals. Our work opens previously unexplored pathways for elastodynamic wave control and energy storage, which may be translated to other phononic and photonic systems of technological relevance.

Pharmacological inhibition of protein kinase CK2 reverts the multidrug resistance phenotype of a CEM cell line characterized by high CK2 level.

Protein kinase CK2 is an ubiquitous and constitutively active kinase, which phosphorylates many cellular proteins and is implicated in the regulation of cell survival, proliferation and transformation. We investigated its possible involvement in the multidrug resistance phenotype (MDR) by analysing its level in two variants of CEM cells, namely S-CEM and R-CEM, normally sensitive or resistant to chemical apoptosis, respectively. We found that, while the CK2 regulatory subunit beta was equally expressed in the two cell variants, CK2alpha catalytic subunit was higher in R-CEM and this was accompanied by a higher phosphorylation of endogenous protein substrates. Pharmacological downregulation of CK2 activity by a panel of specific inhibitors, or knockdown of CK2alpha expression by RNA interference, were able to induce cell death in R-CEM. CK2 inhibitors could promote an increased uptake of chemotherapeutic drugs inside the cells and sensitize them to drug-induced apoptosis in a co-operative manner. CK2 blockade was also effective in inducing cell death of a different MDR line (U2OS). We therefore conclude that inhibition of CK2 can be considered as a promising tool to revert the MDR phenotype.

Therapeutic targeting of CK2 in acute and chronic leukemias.

CK2 is a ubiquitously expressed, constitutively active Ser/Thr protein kinase, which is considered the most pleiotropic protein kinase in the human kinome. Such a pleiotropy explains the involvement of CK2 in many cellular events. However, its predominant roles are stimulation of cell growth and prevention of apoptosis. High levels of CK2 messenger RNA and protein are associated with CK2 pathological functions in human cancers. Over the last decade, basic and translational studies have provided evidence of CK2 as a pivotal molecule driving the growth of different blood malignancies. CK2 overexpression has been demonstrated in nearly all the types of hematological cancers, including acute and chronic leukemias, where CK2 is a key regulator of signaling networks critical for cell proliferation, survival and drug resistance. The findings that emerged from these studies suggest that CK2 could be a valuable therapeutic target in leukemias and supported the initiation of clinical trials using CK2 antagonists. In this review, we summarize the recent advances on the understanding of the signaling pathways involved in CK2 inhibition-mediated effects with a particular emphasis on the combinatorial use of CK2 inhibitors as novel therapeutic strategies for treating both acute and chronic leukemia patients.

Protein kinase CK2 phosphorylates and upregulates Akt/PKB.

Treatment of Jurkat cells with specific inhibitors of protein kinase CK2 induces apoptosis. Here we provide evidence that the anti-apoptotic effect of CK2 can be at least partially mediated by upregulation of the Akt/PKB pathway. Such a conclusion is based on the following observations: (1) inhibition of CK2 by cell treatment with two structurally unrelated CK2 inhibitors induces downregulation of Akt/PKB, as judged from decreased phosphorylation of its physiological targets, and immunoprecipitate kinase assay; (2) similar results are observed upon reduction of CK2 catalytic subunit by the RNA-interference technique; (3) Akt/PKB Ser129 is phosphorylated by CK2 in vitro and in vivo; (4) such a phosphorylation of activated Akt/PKB correlates with a further increase in catalytic activity. These data disclose an unanticipated mechanism by which constitutive phosphorylation by CK2 may be required for maximal activation of Akt/PKB.

Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.

The antioxidant butylated hydroxytoluene stimulates platelet protein kinase C and inhibits subsequent protein phosphorylation induced by thrombin.

The phenolic antioxidant 2,6-bis(1,1-dimethyl ethyl)-4-methylphenol (BHT) evokes a transient phosphorylation of two platelet proteins of Mr 20,000 and 47,000 that are well-known substrates of protein kinase C (PKC) and, similarly to phorbol esters, a slight but persistent phosphorylation of a protein of Mr 26,000. These effects are observed both in the presence and in the absence of extracellular calcium, but are abolished in the presence of the protein kinase C inhibitor staurosporine. The phosphorylation of the 47 kDa protein takes place mostly at the serine and, to a lesser extent, at threonine residues. BHT induces an increased binding of tritiated phorbol dibutyrate to platelets indicating a PKC translocation from cytosol to plasma membrane. Addition of BHT (20 microM) a few min prior to thrombin causes inhibition of both agonist-evoked protein phosphorylation and increase in the Ca2+ concentration, the latter inhibition being counteracted by staurosporine. The inhibitory effect lasts for several minutes even after removal of BHT from the cellular suspending medium. Similar results are obtained with nordihydroguaiaretic acid, whereas 2- and 3-tert-butyl-4-methoxyphenol (BHA) produce only slight effects. BHT activates the protein kinase C purified from pig brain in a concentration-dependent manner (up to 200 microM), whereas it does not affect the activity of other purified protein kinases such as type 1 and 2 casein kinases, type II A, II B and III tyrosine protein kinases from rat spleen and the catalytic subunit of cyclic AMP-dependent protein kinase. It is concluded that, similarly to diacylglycerols and phorbol esters, these phenolic antioxidants activate the protein kinase C, which in turn desensitizes platelets towards subsequent phospholipase C activation.

Cyclic GMP and nitroprusside inhibit the activation of human platelets by fluoroaluminate.

Sodium nitroprusside, an activator of the soluble guanylate cyclase, inhibits the intracellular Ca2+ mobilization, ATP secretion and aggregation of human platelets evoked by fluoroaluminate. Similar results are obtained with 8-bromo-cyclic GMP (8-Br-cGMP). Both nitroprusside and 8-Br-cGMP inhibit the protein kinase C-dependent phosphorylation of the 47 and 20 kDa proteins induced by fluoroaluminate, but not by the protein kinase C activators phorbol ester and diacylglycerol. Since fluoroaluminate interacts directly with a G protein, the present results suggest that the cGMP interferes with platelet activation at the level of G protein-phospholipase C.

Platelet responses promoted by the activation of protein kinase C or the increase of cytosolic Ca2+ are potentiated by adrenaline. Effects of cAMP and staurosporine.

We studied the action of the alpha 2 adrenergic agonist adrenaline on the platelet responses evoked by the activation of protein kinase C or by the ionophore induced increase of cytosolic Ca2+. Both the phorbol ester and ionomycin-induced aggregation are strongly potentiated by adrenaline which per se does not behave as an activating agonist. The potentiation by adrenaline is observed both when added before and after the aggregating agent; in the latter case the effect increases on increasing the delay of adrenaline addition. Adrenaline also reverses the inhibition by cAMP of the PMA (or ionomycin) induced aggregation. It also has a strong potentiating effect (over 100%) on the phorbol ester induced ATP secretion and a weaker effect on the secretion induced by ionomycin. The effect on secretion is visible only when adrenaline is added prior to the stimulus. The inhibition by cAMP of the PMA or ionomycin induced secretion is also counteracted by adrenaline. In no case adrenaline modifies the pattern of platelet phosphoproteins. Ionomycin induces some platelet aggregation also in the presence of the protein kinase inhibitor staurosporine; also this phosphoprotein independent aggregation is strongly stimulated by adrenaline.

Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa.

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.

Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by Src homology 2 domains of the Src family.

Protein tyrosine kinase p72syk purified from rat spleen has been assayed for its ability to phosphorylate a number of peptide substrates derived from naturally occurring phospho-acceptor sites. The phosphorylation efficiency is extremely variable, depending on the peptide sequence, with Km values in the 3-1500 microM range. The by far best peptide substrates, with Km values of 3 and 4 microM are those reproducing the phospho-acceptor sites of Vav and HS1 proteins, respectively. These sites include multiple acidic residues flanking tyrosine on both sides and they also display the consensus sequences (YEDL and YEEV) preferred by the SH2 domains of the Src family. Alteration of this consensus in the HS1 peptide, by replacing either the glutamic acid or valine, also reduces the phosphorylation efficiency by p72syk. Also the replacement of acidic residues at position -1 and, to a lesser extent at positions -3 and -4 (but not at positions +3 and +5) are detrimental. These observations may suggest a role of p72syk in the recruitment of ligands/substrates for the Src family enzymes. We also show that the HS1 peptide can be used for the specific monitoring of p72syk since neither the two Src-related c-Fgr and Lyn kinases (needing a hydrophobic instead of acidic residue at position -1) nor CSK appreciably phosphorylate it.

Hematopoietic lineage cell specific protein 1 associates with and down-regulates protein kinase CK2.

The catalytic (alpha) subunit of protein kinase CK2 and the hematopoietic specific protein 1 (HS1) display opposite effects on Ha-ras induced fibroblast transformation, by enhancing and counteracting it, respectively. Here we show the occurrence of physical association between HS1 and CK2alpha as judged from both far Western blot and plasmon resonance (BIAcore) analysis. Association of HS1 with CK2alpha is drastically reduced by the deletion of the HS1 C-terminal region (403-486) containing an SH3 domain. HS1, but not its deletion mutant HS1 Delta324-393, lacking a sequence similar to an acidic stretch of the regulatory beta-subunit of CK2, inhibits calmodulin phosphorylation by CK2alpha. These data indicate that HS1 physically interacts with CK2alpha and down-regulates its activity by a mechanism similar to the beta-subunit.

The carboxy-terminal domain of Grp94 binds to protein kinase CK2 alpha but not to CK2 holoenzyme.

Surface plasmon resonance analysis shows that the carboxy-terminal domain of Grp94 (Grp94-CT, residues 518-803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2 alpha) under non-stressed conditions. A K(D) of 4 x 10(-7) was determined for this binding. Heparin competed with Grp94-CT for binding to CK2 alpha. CK2 beta also inhibited the binding of Grp94-CT to CK2 alpha, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94-CT. The use of CK2 alpha mutants made it possible to map the Grp94-CT binding site to the four lysine stretch (residues 74-77) present in helix C of CK2 alpha. Grp94-CT stimulated the activity of CK2 alpha wild-type but was ineffective on the CK2 alpha K74-77A mutant.

Selectivity of 4,5,6,7-tetrabromobenzotriazole, an ATP site-directed inhibitor of protein kinase CK2 ('casein kinase-2').

The specificity of 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an ATP/GTP competitive inhibitor of protein kinase casein kinase-2 (CK2), has been examined against a panel of 33 protein kinases, either Ser/Thr- or Tyr-specific. In the presence of 10 microM TBB (and 100 microM ATP) only CK2 was drastically inhibited (>85%) whereas three kinases (phosphorylase kinase, glycogen synthase kinase 3 beta and cyclin-dependent kinase 2/cyclin A) underwent moderate inhibition, with IC(50) values one--two orders of magnitude higher than CK2 (IC(50)=0.9 microM). TBB also inhibits endogenous CK2 in cultured Jurkat cells. A CK2 mutant in which Val66 has been replaced by alanine is much less susceptible to inhibition by TBB as well as by another ATP competitive inhibitor, emodin. These data show that TBB is a quite selective inhibitor of CK2, that can be used in cell-based assays.

Phosphatidylinositol 3-kinase is recruited to a specific site in the activated IL-1 receptor I.

Interleukin 1 (IL-1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF-kappaB. The IL-1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C-terminal portion of the type I IL-1 receptor (IL-IRI) is essential for IL-1 signalling and for IL-1 dependent activation of NF-kappaB. This portion contains a putative phosphatidylinositol 3-kinase (PI 3-kinase) binding domain (Tyr-E-X-Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3-kinase in IL-1 signalling. Here we report evidence that PI 3-kinase is recruited by the activated IL-IRI, causing rapid and transient activation of PI 3-kinase. We also show that the receptor is tyrosine phosphorylated in response to IL-1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3-kinase activation by IL-1. Our results indicate that PI 3-kinase is likely to be an important mediator of some IL-1 effects, providing docking sites for additional signalling molecules.

Purification and characterization of two casein kinases from ejaculated bovine spermatozoa.

Two protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19 microM). Enzyme activity was maximal in the presence of 10 mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250 mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodies raised against recombinant alpha, but not beta, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 microM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30 mM), and NaCl (20-400 mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.

A procedure allowing measurement of cytosolic Ca2+ in rat platelets. Inhibition of a plasma lipoprotein on fura 2-AM loading.

Loading of the fluorescent Ca2+ probe fura 2 in rat platelets is highly inhibited by a plasmatic factor that is removed by gel filtration through a Sepharose C-2B column. Rat plasma also inhibits fura 2 loading in human platelets. The inhibitory effect is abolished by perchloric acid-deproteinization or heat denaturation of plasma suggesting a proteic nature of the inhibitory compound. Indeed the 10,000 x g supernatant of the heat denaturated plasma shows a positive effect on fura 2 accumulation, most likely by partially inhibiting its cellular effux. These effects are only negligibly shown by the corresponding fractions of human plasma. Results obtained by fractionation of rat plasma proteins by means of ion exchange DEAE Sepharose C-6B chromatography and ultracentrifugation through high density saline solutions indicate that the inhibition of cellular fura 2 loading is due to the HDL fraction of rat plasma.

Psoralen-fatty acid cycloadducts activate protein kinase C (PKC) in human platelets.

The C4-photocycloadduct psoralen-linolenic acid activates protein kinase C (PKC), in human platelets, which in turn phosphorylates 47 kDa protein, representing its major substrate in these cells. This behaviour is similar to that shown by diacylglycerol, a main second messenger responsible of various biological effects, e.g. of melanogenesis.

Protein kinase CK2 in hematologic malignancies: reliance on a pivotal cell survival regulator by oncogenic signaling pathways.

CK2 is a multitask kinase whose role is essential for a countless number of cellular processes, many of which are critical for blood cell development. A prevailing task for this kinase rests on counteracting programmed cell death triggered by multiple stimuli. CK2 is overexpressed in many solid tumors and in vivo mouse models have proven its tumorigenic potential. Recent data have suggested that CK2 may also have a significant role in the pathogenesis of hematopoietic tumors, such as multiple myeloma, chronic lymphocytic leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia and chronic myeloproliferative neoplasms. CK2 regulates hematopoiesis-associated signaling pathways and seems to reinforce biochemical cascades indispensable for tumor growth, proliferation and resistance to conventional and novel cytotoxic agents. Although its activity is multifold, recent evidence supports the rationale of CK2 inhibition as a therapeutic strategy in solid and hematological tumors and phase-I clinical trials are in progress to test the efficacy of this innovative therapeutic approach. In this review, we will summarize the data supporting CK2 as an oncogenic kinase in blood tumors and we will describe some critical signaling pathways, whose regulation by this protein kinase may be implicated in tumorigenesis.