RESUMO
Dissociated GCs show a separation between anti-inflammatory effects and certain side effects. This renders them as attractive compounds with better effect/side-effect profile as promising drug candidates and tool compounds for analyzing the molecular mechanisms of single side effects. A number of the GC-mediated side effects (e.g., osteoporosis, skin atrophy) are regulated in a very complex manner and use more than one molecular mechanism of the GR. Thus, theoretical predictions about the behavior of selective GR agonists regarding these effects are very difficult to make. Investigations of SEGRA compounds in relevant animal models will be the only way to get this important information. By availability of these tool compounds we now are in the advantageous situation to test them in vivo and to learn more about the possibilities and even the limitations of the selective GR agonists. Considering that the compounds have a non-steroidal structure, i.e., totally unrelated to steroids or other hormones at all, displaying only partially the molecular effects of GCs and are dissociated in their clinical profile, they should not be considered as GCs. Therefore, we introduced the term selective glucocorticoid receptor agonists (SEGRAs). These SEGRAs seem to represent a useful novel therapeutic modality which may complement existing therapeutic principles for the topical and especially the systemic treatment of inflammatory diseases. In summary, we and others are convinced that dissociated GCs are therapeutic compounds that exert many of the anti-inflammatory and immunosuppressive effects of standard GCs, while their potential to induce side effects is reduced. Whereas the in vitro dissociated profile of other compound classes (Belvisi et al. 2001) was not translated into a separation between anti-inflammatory activity and the induction of side effects in in vivo models, we could demonstrate this for the SEGRA compounds. Regarding the diversity of molecular mechanisms involved in mediating the complex side effects of GCs, it might be that only some of these unwanted effects can be reduced. However, as GCs are one of the most important anti-inflammatory therapeutics in the treatment of severe and chronic inflammatory diseases, even a partial reduction of side effect induction would be a great advantage for many patients.
Assuntos
Anti-Inflamatórios/farmacologia , Administração Tópica , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/efeitos adversos , Glucocorticoides/administração & dosagem , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacologia , Humanos , Camundongos , Receptores de Glucocorticoides/agonistasRESUMO
Mature A- and B-type lamins differ in the extent to which they interact with the nuclear membrane and thus represent an interesting model for studying the role of isoprenylation and carboxyl-methylation in membrane attachment. Both A- and B-type lamins are isoprenylated and carboxyl-methylated shortly after synthesis, but A-type lamins undergo a further proteolytic cleavage which results in the loss of the hydrophobically modified C terminus. Here, we have constructed mutants of chicken lamin A that differ in their abilities to serve as substrates for different post-translational processing events occurring at the C terminus of the wild-type precursor. In addition to studying full-length proteins, we have analyzed C-terminal end domains of lamin A, either alone or after fusion to reporter proteins. Mutant proteins were expressed in mammalian cells, and their membrane association was analyzed by immunofluorescence microscopy and subcellular fractionation. Our results provide information on the substrate specificity and subcellular localization of the lamin-A-specific protease. Moreover, they indicate that hydrophobic modifications of the C-terminal end domains account for the differential membrane-binding properties of A- and B-type lamins. Thus, some of the integral membrane proteins implicated in anchoring B-type lamins to the membrane may function as receptors for the isoprenylated and carboxyl-methylated C terminus.
Assuntos
Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Carcinoma Embrionário/patologia , Galinhas , Cromatina/metabolismo , Endopeptidases/metabolismo , Células HeLa , Humanos , Lamina Tipo A , Laminas , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Ligação Proteica , Precursores de Proteínas/genética , Prenilação de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/química , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
The nuclear lamina is a karyoskeletal structure at the nucleoplasmic surface of the inner nuclear membrane. Its assembly state is regulated by phosphorylation of the intermediate filament type lamin proteins. Strong evidence has been obtained for a causal link between phosphorylation of lamins by the p34cdc2 protein kinase and disassembly of the nuclear lamina during mitosis. In contrast, no information is currently available on the role of lamin phosphorylation during interphase of the cell cycle. Here, we have identified four protein kinase C phosphorylation sites in purified chicken lamin B2 as serines 400, 404, 410, and 411. In vivo, the tryptic peptide containing serines 400 and 404 is phosphorylated throughout interphase, whereas serines 410 and 411 become phosphorylated specifically in response to activation of protein kinase C by phorbol ester. Prompted by the close proximity of serines 410/411 to the nuclear localization signal of lamin B2, we have studied the influence of phosphorylation of these residues on nuclear transport. Using an in vitro assay, we show that phosphorylation of lamin B2 by protein kinase C strongly inhibits transport to the nucleus. Moreover, phorbol ester treatment of intact cells leads to a substantial reduction of the rate of nuclear import of newly synthesized lamin B2 in vivo. These findings have implications for the dynamic structure of the nuclear lamina, and they suggest that the modulation of nuclear transport rates by cytoplasmic phosphorylation may represent a general mechanism for regulating nuclear activities.
Assuntos
Núcleo Celular/metabolismo , Lamina Tipo B , Proteínas Nucleares/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Células HeLa , Humanos , Interfase , Laminas , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Mapeamento de Peptídeos , Fosfopeptídeos/isolamento & purificação , Fosforilação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Serina , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.
Assuntos
Proteína Quinase C/metabolismo , Proteínas Quinases/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Bufo marinus , Patos , Ativação Enzimática , Técnicas In Vitro , Rim/enzimologia , Microssomos/enzimologia , Oócitos/enzimologia , Fosforilação , Glândula de Sal/enzimologia , Xenopus laevisRESUMO
Using a polyclonal antibody, a cDNA clone coding for part of mouse vimentin was identified in a lambda gt11 expression library. DNA from this clone was used to screen a genomic library from Ehrlich Ascites Tumor cells for the mouse vimentin gene. A clone was found which contained the whole coding sequence and a large part of the 5'- and 3'-untranslated sequences. It was used to prepare a construct equivalent to a full-length cDNA clone. Extensive homologies to the vimentin sequence from other species were found for the coding and 3'-untranslated sequences and the promoter region.
Assuntos
Éxons , Vimentina/genética , Animais , Sequência de Bases , Genes , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
Pulsed field gel electrophoresis allows not only the separation of very large DNA molecules (up to 10 megabase pairs) but also gives an enhanced resolution in separations of DNA in the size range of 10-100 kilobase pairs (kbp). For this application, rapid alternation of the electrical field polarity is required. Here we describe equipment for the delivery of short switching pulses that is easy and inexpensive to build and is controlled by a standard microcomputer. It has proved to be useful in the separation of lambda DNA and its fragments. Parameters for enhanced separation of 23- and 48-kbp DNA molecules at high voltage gradients (15 V/cm) are presented and shown to provide superior resolution when compared to those for conventional electrophoresis at both high and low voltage gradients.