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Previously, we reported significantly reduced pain and improved mobility persisting for 6 months after kyphoplasty of chronically painful osteoporotic vertebral fractures in the first prospective controlled trial. Since improvement of spinal biomechanics by restoration of vertebral morphology may affect the incidence of fracture, long-term clinical benefit and thereby cost-effectiveness, here we extend our previous work to assess occurrence of new vertebral fractures and clinical parameters 1 year after kyphoplasty compared with a conservatively treated control group. Sixty patients with osteoporotic vertebral fractures due to primary osteoporosis were included: 40 patients were treated with kyphoplasty, 20 served as controls. All patients received standard medical treatment. Morphological characteristics, new vertebral fractures, pain (visual analog scale), physical function [European Vertebral Osteoporosis Study (EVOS) score] (range 0–100 each) and back-pain-related doctors’ visits were re-assessed 12 months after kyphoplasty. There were significantly fewer patients with new vertebral fractures of the thoracic and lumbar spine, after 12-months, in the kyphoplasty group than in the control group ( P=0.0084). Pain scores improved from 26.2 to 44.4 in the kyphoplasty group and changed from 33.6 to 34.3 in the control group ( P=0.008). Kyphoplasty treated patients required a mean of 5.3 back-pain-related doctors’ visits per patient compared with 11.6 in the control group during 12 months follow-up ( P=0.006). Kyphoplasty as an addition to medical treatment and when performed in appropriately selected patients by an interdisciplinary team persistently improves pain and reduces occurrence of new vertebral fractures and healthcare utilization for at least 12 months in individuals with primary osteoporosis. [ABSTRACT FROM AUTHOR]

Little is known of how protein kinase CK2 genes are regulated, and it is unclear whether there are mechanisms of transcriptional coordination. Response elements present in the promoter sequences of the human catalytic (CK2α) and regulatory (CK2β) subunit genes have been examined for the significance in transcriptional control using reporter gene assays, electrophoretic mobility shift assays, site-directed mutagenesis, ectopic protein expressions, and transcript assessments. Most strikingly, in both promoters the regions of highest transcriptional activity contain two adjoining, completely identical and conserved Ets1 response elements, and both the mutation of motifs and the overexpression of Ets1 affect significantly transcriptional activity. Also in common are Sp1 response elements that cooperate with Ets1, and Sp1 is phosphorylatable by CK2 holoenzyme but not by individual CK2α, the phosphorylation negatively affecting DNA binding. CK2α and CK2β transcript levels and stoichiometries of mRNA species turned out quite constant in cultured cells despite progressing through various stages of proliferation and differentiation. The data seem to indicate transcriptional coordination of the human genes encoding CK2α and CK2β based on an Ets1 double motif common to both genes cooperating with Sp1 motifs and amenable to negative feedback control by the gene products which, following complexation into CK2 holoenzyme, could phosphorylate Sp1 (and Ets1?) and thus downregulate transcription and contribute to the observed constant cellular CK2α and CK2β transcripts situation. [ABSTRACT FROM AUTHOR]

Protein kinase CK2, a tetramer composed of two catalytically active (CK2α isoforms) and two regulatory (CK2α isoforms) subunits, is suspected to have, among others, a role in gene transcription. To identify the genes targeted by CK2, the transcriptional effect of silencing the CK2 subunit genes in Saccharomyces cerevisiae (CK2α isoform genes: CKA1 and CKA2; CK2α isoform genes: CKB1 and CKB2) was examined using genome-wide expression array analysis (oligonucleotide array chips). Silencing did not influence the overwhelming majority (5801) of the over six thousand open reading frames composing the yeast genome. Cells knocked-out for both CKA1 and CKA2 and plasmid-rescued by Cka1 affected specifically at 2-fold discrimination level the transcription of 57 genes, and when rescued by Cka2, the transcription of 118 genes. In CKB1/CKB2 double knock-outs, transcription of 54 genes was specifically altered. Interestingly, aside overlaps between the gene spectra affected by CKA1 and CKA2 silencing, there were overlaps also between those influenced by CK2α and CK2α isoform silencing. The data indicate a distinct role of CK2 in gene transcription control, identify specific functional differences between the two catalytic subunits in gene targeting, and reveal independent effects by the regulatory subunits. [ABSTRACT FROM AUTHOR]

Chemical crosslinking and analysis of CNBr-digested fusion products by immunoblotting with sequence-specific antibodies identifies an interaction between positions 55-70 of subunit β (β55-70) and 65-80 of subunit α (α65-80). This has been supported by crosslinking of subunits with peptides α65-80 and β55-70, by binding of subunits to immobilized peptides, and by the hindrance of coprecipitation with peptide-raised antibodies (anti-α65-80; anti-β55-70). Functionally, β55-70 is a negative regulatory region for the kinase activity of subunit α. The opposite, stimulatory property of subunit β has been assigned to its C-terminal part. Subdivision of peptide β155-181, that has stimulatory effect, into overlapping peptides and assaying for a binding and binding competition revealed a tight physical contact at β162-175. This region, however, is non-stimulatory indicating binding a necessary but not sufficient quality for stimulation. A contact might exist to regions surrounding C147 and/or C220 at subunit a as indicated by crosslinking and peptide competition. The crosslinking data also confirm a β-β contact in CK2 holoenzyme. Effects by non-ionic detergents show hydrophobic interactions to play an important role in catalytic activity adjustment. [ABSTRACT FROM AUTHOR]

Two CK2α loci are present in the human genome. First, locus 20p13, that contains the CK2α coding gene. It spans around 70 kb, is composed of 13 exons and shows homology to the respective gene in the nematode Caenorhabditis elegans. The translation start site is located in the second exon, the stop codon in exon 13. Two transcription start sites were identified, the further 5′ located site defines position 1 of the gene, the second site is located at position 50, respectively. The promoter region shows characteristics of a so-called house keeping gene: A high GC content, lack of a TATA-box and presence of several GC-boxes. By reporter gene assays, the promoter region of the CK2α gene could be located between position -256 and 144 relative to the first transcription start site. In the 3′ noncoding region of the CK2α gene, six polyadenylation signals were identified. As indicated by Northern blot analysis and by comparison with expressed sequence tags from the EMBL databank, the most 3′ located, active polyadenylation signal seems to be the fourth defining the end of the CK2α gene. The second CK2α locus is at 11p15. It contains a processed pseudogene, which shows all typical features of a processed sequence, such as absence of introns, short poly-A tail and direct flanking repeats. Interestingly, it contains a complete open reading frame and has potential promoter elements in its 5′ region. Nevertheless, no promoter activity could be detected in reporter gene assays. [ABSTRACT FROM AUTHOR]

BTF3, initially discovered as a factor required for transcription inititation of RNA polymerase II, is expressed in two isoforms, termed a and b. BTF3b, the transcriptionally inactive isoform, was identified as an interaction partner of protein kinase CK2 subunit β employing the interaction trap system for screening of a HeLa cDNA fusion library. We report here on the interaction between the other isoform, BTF3a, and protein kinase CK2. The complete cDNA of BTF3a was cloned by RT-PCR and used for analysis in the two-hybrid system with a three-reporter yeast strain. Interaction of BTF3a with CK2 subunits α, α′ or β was detectable by one of three reporters, whereas the CK2β- BTF3a interaction was activating two reporters. It was also shown that BTF3a is phosphorylatedin vitro by the α2β2 holoenzyme, but not by α or α′ alone, indicating the requirement of β for substrate recognition. Immunoprecipitations of GST-fused BTF3a carried out in vitro resulted in co-precipitation of β. Similarly, GST-BTF3a, but not GST alone isolated with glutathione agarose beads from buffer containing recombinant CK2 subunits was found complexed with α and β, likely representing α2β2 holoenzyme. The data show a weak, nevertheless specific interaction of protein kinase CK2 via subunit β with the putative transcription factor BTF3a in vitro and in vivo, and a role of BTF3a as a potential new substrate for CK2. [ABSTRACT FROM AUTHOR]

The gene-inducing property of CK2α, a Ser/Thr protein kinase that appears normally to be complexed to a CK2β protein controlling activity and substrate selectivity, has been unclear. We show here that CK2α induces in human JEG-3 cells the expression of Aromatase, an estrogen-synthesis key enzyme, which is regulated at transcriptional level. Electrophoretic mobility shift assays indicate that CK2α binds to the Aromatase gene promoter. To test for CK2α's transactivating ability as a DNA-binding protein, a CK2α binding site was cloned in front of indicator genes. The constructs were used to transform a yeast-based one-hybrid system. Overexpression of activation-domain fused CK2α in this system, i.e., CK2α in its native configuration, failed to activate the transcription machinery. The data indicate CK2α to affect gene expression at the level of transcription via an indirect as yet unknown mechanism rather than directly as a DNA-binding transcription-activating protein. [ABSTRACT FROM AUTHOR]

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2α′ was more resistant to these proteases than CK2α. When these proteases were assayed on the reconstituted (α2β2 holoenzyme, a 37 kDa α-band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2α deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2α. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells. [ABSTRACT FROM AUTHOR]

Hepatic microsomal monooxygenase was reconstituted by incorporating cytochrome P-450 and NADPH-cytochrome P-450 reductase, which had been purified from phenobarbital-pretreated rabbit liver microsomes, into phospholipid liposomal membranes. The NADPH-dependent monooxygenase activity of the reconstituted system was found to be dependent on the phospholipid-to-protein ratio, i.e., the two-dimensional concentration of the two proteins on the plane of the membranes. A similar concentration dependence was also observed in the cytochrome b and NADH-cytochrome b system, which had been incorporated into liposomal membranes. The diffusion process of the proteins in the membrane, therefore, plays an important role in the monooxygenase system. When the fluidity of the membrane was changed by utilizing a synthetic dimyristoylphosphatidylcholine, which shows a well-defined gel to liquid crystalline phase transition, the activation energy of the monooxygenase reaction was changed at around the phase transition temperature, suggesting a conformational change of cytochrome P-450 caused by the fluidity change of the membrane. The incorporation of P-450 into liposomes was also found to affect the binding of substrates to cytochrome P-450. The decrease in the apparent dissociation constant of substrates upon incorporation into membranes suggests that the lipid membrane acts as a pool for hydrophobic substrates, which are concentrated in the lipid phase, and that cytochrome P-450 takes substrates directly from the membrane phase. Phospholipid membranes, therefore, play very important roles in various phases of the reaction of cytochrome P-450-dependent monooxygenase. [ABSTRACT FROM AUTHOR]

Arylhydrocarbons are inducers of monooxygenase activity in epithelial cells in culture (Hela) whereas diterpene esters are not. This is one more example of a specific biochemical difference - here in gene activation - exhibited by tumor initiators and tumor promoters of the classes of compounds assayed. [ABSTRACT FROM AUTHOR]