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Introduction: Osteoporosis and osteoporotic fractures typically affect postmenopausal women, and osteoporotic fractures significantly increase disability, morbidity and mortality. Several anti-osteoporotic agents are available and have been shown to effectively reduce the incidence of low-energy osteoporotic fractures. However, the post-osteoporotic fracture treatment rate remains low. The purpose of the present study was to follow up on patients with a recent hip fracture with primary focus on anti-osteoporotic treatment and dual-energy X-ray absorptiometry (DXA)., Methods: We included patients ≥ 65 years of age admitted to a department of orthopaedic surgery in Denmark from 1 June 2019 to 30 May 2020., Results: In this period, 570 patients ≥ 65 years were treated for a hip fracture. A total of 16.7% of the patients received anti-osteoporotic treatment at follow-up and 6.5% initiated anti-osteoporotic treatment or had a relevant change in anti-osteoporotic treatment. Only 9.8% had a DXA after their fracture; and among this group, 48% received anti-osteoporotic treatment., Conclusion: The majority of patients with a recent low-energy hip fracture did not receive a DXA, did not have a relevant follow-up or received any anti-osteoporotic treatment. The problem is global and needs to be addressed. Starting treatment with anti-osteoporotic medicine before discharge from the orthopaedic department and referring the patient to a DXA at the same time may be part of the solution., Funding: none., Trail Registration: not relevant., (Articles published in the DMJ are “open access”. This means that the articles are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.)

Delirium is common in hospitalized older adults. The condition is frequently not recognized, or managed appropriately, and has a poor prognosis. This review finds that a proactive multicomponent interdisciplinary non-farmacological approach can reduce incidence. Delirium is managed by identification of the condition, accurate diagnosis and treatment of the causes, and all other correctable contributing factors, using nonpharmacologic approaches. In some cases, and if required for patient safety, low doses of high-potency antipsychotic agents can be used, in lowest possible dose and for the shortest possible time.

The atypical protein kinase C (PKC) member PKC-zeta has been implicated in several signal transduction pathways regulating differentiation, proliferation or apoptosis of mammalian cells. We report here the identification of a cytoplasmic and membrane-associated protein that we name zeta-interacting protein (ZIP) and that interacts with the regulatory domain of PKC-zeta but not classic PKCs. The structural motifs in ZIP include a recently defined ZZ zinc finger as a potential protein binding module, two PEST sequences and a novel putative protein binding motif with the consensus sequence YXDEDX5SDEE/D. ZIP binds to the pseudosubstrate region in the regulatory domain of PKC-zeta and is phosphorylated by PKC-zeta in vitro. ZIP dimerizes via the same region that promotes binding to PKC-zeta suggesting a competitive situation between ZIP:ZIP and ZIP:PKC-zeta complexes. In the absence of PKC-zeta proper subcellular localization of ZIP is impaired and we show that intracellular targeting of ZIP is dependent on a balanced interaction with PKC-zeta. Taking into account the recent isolation of ZIP by others in different contexts we propose that ZIP may function as a scaffold protein linking PKC-zeta to protein tyrosine kinases and cytokine receptors.

Cross-linking of the antigen receptor on lymphocytes by antigens or antibodies to the receptor results in activation of enzymes of the protein kinase C (PKC) family. Mice homozygous for a targeted disruption of the gene encoding the PKC-betaI and PKC-betaII isoforms develop an immunodeficiency characterized by impaired humoral immune responses and reduced cellular responses of B cells, which is similar to X-linked immunodeficiency in mice. Thus PKC-betaI and PKC-betaII play an important role in B cell activation and may be functionally linked to Bruton's tyrosine kinase in antigen receptor-mediated signal transduction.

The extracellular domain of the human interleukin-6 (IL-6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus-infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, using IL-6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 x 10(4) l.mol-1.cm-1 was calculated from the ultraviolet spectrum of the soluble IL-6 receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with peptide N-glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41-44 kDa. As expected, the soluble human IL-6 receptor bound human 125I-labeled IL-6 with low affinity (Kd = 500 pM). Furthermore, the binding of soluble human IL-6 receptor to immobilized IL-6 was studied using real-time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL-6 (HepG2-IL-6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL-6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1-67 and 68-143 of the soluble IL-6 receptor, respectively. The plasma clearance of soluble 125I-labeled IL-6 receptor in the absence and presence of IL-6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL-6 receptor/IL-6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble 125I-labeled IL-6 receptor, as well as complexes with IL-6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys. Subsequently, the radioactivity appeared in the gut content.

Considerable effort has been devoted to identifying critical steps in mitogenic signal transduction pathways. Recently, the atypical PKC zeta isoform has attracted great interest since it has been reported to induce GVBD in Xenopus oocytes and transformation of NIH3T3 fibroblasts, two processes closely linked with the regulation of cell division. Furthermore, PKC zeta has been proposed as an essential effector for ras-p21 function and therefore may be an essential component of the signalling pathway(s) activated by mitogens. In this study we have analysed the responses induced in Xenopus oocytes after microinjection of purified recombinant PKC zeta protein. Microinjection of PKC zeta induced the early activation of MPF which precedes GVBD and also induced the activation of MAP kinase and S6 kinase II. The activation of MPF, MAP kinase and S6 kinase II by PKC zeta was sensitive to cycloheximide, while induction of GVBD was independent of protein synthesis. These results indicate that PKC zeta induces the activation of at least two pathways, only one of them leading to the activation of MAP kinase. By contrast, neither the induction of GVBD nor the activation of MPF, MAPK and S6 kinase II induced by the ras-p21 protein were dependent on protein synthesis. Thus, the comparison of these responses suggests that PKC zeta most likely does not mediate the ras-induced signal transduction pathway in Xenopus laevis oocytes.

A proportion of the microtubule-associated protein, tau, is in an elevated state of phosphorylation in foetal and adult brain whereas all of the tau in paired helical filaments, which are characteristic of Alzheimer's disease is hyperphosphorylated; it is important therefore to elucidate the mechanisms that regulate tau phosphorylation. Here we describe results that show that although MAP kinase can hyperphosphorylate tau in vitro, activation of MAP kinase in transformed fibroblasts does not result in hyperphosphorylation of transfected tau, whereas glycogen synthase kinase-3 beta (GSK-3 beta) when co-transfected with tau does result in tau hyperphosphorylation. The findings imply that GSK-3 beta may be a stronger candidate than MAP kinase for inducing tau hyperphosphorylation in vivo.

The Mos protein kinase is a serine-/threonine-specific protein kinase with a crucial role in meiotic cell divisions in vertebrates. Several oncogenic derivatives of the c-Mos protein have been discovered in murine retroviruses. These proteins have acquired mutations and exhibit different degrees of protein kinase activity in vitro. In an attempt to understand the factors governing Mos protein kinase activity we have compared the kinase activities of the wild-type c-Mos protein and two v-Mos proteins (strain HT1 and MSV124) after expression in insect cells. Only the 124 v-Mos protein showed kinase activity in vitro as measured by autophosphorylation, vimentin phosphorylation or by phosphorylation and activation of MAP kinase kinase. By domain swapping and site-directed mutagenesis we identified a single point mutation in the 124 v-Mos protein (Arg145-->Gly) which is responsible for its constitutive activity. This residue is located in the alpha-helix C of the kinase domain close to the ATP binding fold and is conserved in all known c-Mos proteins. Introduction of the corresponding mutation into HT1 v-Mos and into murine c-Mos activated both proteins for autophosphorylation, vimentin phosphorylation and for signalling via MAP kinase kinase in vitro. We hypothesize that the Arg145-->Gly mutation found in 124 v-Mos mimicks a conformational change which might be an obligatory step in the activation of c-Mos in vivo.

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3 alpha, GSK-3 beta and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3 alpha and GSK-3 beta can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3., Results: Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3 alpha or GSK-3 beta decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies., Conclusions: Our data indicate that GSK-3 alpha and/or GSK-3 beta, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.

The phorbol ester PMA/TPA (phorbol 12-myristate 13-acetate) is a potent tumor promoter which mimics distinct intracellular signalling events triggered by activated growth factor receptors, e.g. the activation of MAP kinases. The largest known family of TPA-binding proteins comprise members of the protein kinase C (PKC) family although other TPA-binding proteins outside the PKC family have recently been identified. In this report we addressed the mechanism and the pathway by which TPA induces the activation of MAPkinases. Using recombinant proteins and in vitro phosphorylation reactions we identified the components in the signal transduction pathway from TPA to MAPkinase and we show that the activation of MAPkinase by TPA requires the presence of protein kinase C, c-raf and the MAPkinase activator MEK. We also find that the activation of raf autophosphorylation in vitro correlates with the ability of Raf to signal to MAPkinase. Thus the activation of Raf by PKC apparently can trigger the same signalling pathway as oncogenic Raf or Raf activation by ras in combination with tyrosine phosphorylation.

Treatment of HC11 mouse mammary epithelial cells with the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) leads to cellular differentiation and production of the milk protein beta-casein. The following experimental evidence suggests the involvement of protein kinase C (PKC) in DIP induced signal transduction. Down-regulation of PKC by 12-O-tetradecanoylphorbol-13-acetate or addition of CGP 41251, a selective inhibitor of PKC, inhibited beta-casein protein expression induced by DIP in HC11 cells. This inhibition occurs at the level of transcription, since the DIP mediated activation of a beta-casein promoter-luciferase reporter construct or of mammary gland specific factor (MGF), an essential transcription factor for beta-casein promoter activity, was also inhibited by CGP 41251. Inhibition or down-regulation of PKC reduced the activation of MGF by prolactin as well. PKC-alpha, the only conventional PKC isoform expressed in HC11 cells, is most likely involved in the DIP induced beta-casein expression. (a) Only PKC-alpha and PKC-epsilon are down-regulated by 12-O-tetradecanoylphorbol-13-acetate whereas PKC-delta and PKC-zeta are not. (b) Of the PKC isoforms expressed in HC11 cells, CGP 41251 inhibits PKC-alpha more potently than PKC-delta, PKC-epsilon, and PKC-zeta. The IC50 for the inhibition of beta-casein synthesis, MGF activation, and beta-casein promoter activity by CGP 41251 correlated well with the IC50 of PKC-alpha inhibition. (c) Finally, only PKC-alpha translocated to membrane fractions after DIP or prolactin treatment. Taken together, these data indicate that PKC-alpha plays an important role in the signaling pathway activated by prolactin during beta-casein induction.

Previous in vitro experiments have shown that the phorbol-like diterpenes 12-deoxyphorbol 13-isobutyrate (dPB), and possibly mezerein, have multiple biological target sites which differ from one another in apparent affinity for dPB by 12.5-780 fold and for mezerein by 24-fold. These two compounds are thus very important ligands because of their potential PKC isotype-selectivity. In the present study they were found to have binding affinities differing by a maximum of only 1.6-fold among recombinant protein kinase C (PKC) isotypes alpha, beta 1, beta 2, and gamma (the "A-group") in a [3H]phorbol dibutyrate binding assay. The apparent Ki's were 92-140 nM for dPB and 68-92 nM for mezerein. Our results are consistent with short-term 12-deoxyphorbol ester-induced mouse skin inflammation being mediated at least in part by one or more A-group PKC isotypes. The data also indicate that the pharmacologically distinguishable target sites previously established for mezerein and dPB must include one or more binding sites not found in the A-group of PKC isotypes and that mezerein has a high-affinity, non-A-group target site in brain.

The product of the c-raf-1 proto-oncogene is a cytoplasmic serine/threonine protein kinase that appears to be activated in signal transduction from a variety of cell-surface receptors. The mechanism of c-Raf activation upon stimulation of cell-surface receptors is not clear, but there seem to exist multiple pathways of activation which involve tyrosine and/or serine phosphorylation of the c-Raf protein in vivo. The activated state of Raf is reflected in an increased apparent molecular weight of the Raf protein in sodium dodecyl sulfate-polyacrylamide gels owing to hyperphosphorylation. The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) is one of the agents able to induce this hyperphosphorylation of Raf in vivo, suggesting that protein kinase C (PKC) may be involved in the activation of c-Raf in particular situations. Using recombinant baculoviruses expressing PKC and Raf polypeptides, we show here that conventional PKC types (alpha, beta, gamma) but not novel types (delta, zeta, eta) or the unrelated Mos kinase are able to activate c-Raf in a TPA-dependent manner upon coexpression in insect cells. Direct phosphorylation of the Raf protein with PKC in vitro also enhanced the kinase activity of c-Raf, suggesting that c-Raf acts immediately downstream of PKC in a protein kinase cascade which is triggered by TPA and may lead to transcriptional activation of TPA-inducible genes and tumor promotion.

Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of the group A enzymes. PKC-zeta, a polypeptide of 80 kDa, does not bind phorbol ester and does not phosphorylate these substrates to a significant extent. Consistent with their ability to bind phorbol ester, recombinant PKC-delta and PKC-epsilon are down-regulated in COS cells by prolonged treatment with phorbol ester, whereas PKC-zeta protein levels remain unaltered.