Use of Calcilytic drugs as a pharmacological approach to the treatment and prevention of Alzheimer's Disease, Alzheimer's disease-related disorder, and down's syndrome neuropathies

Use of Calcilytic drugs as a pharmacological approach to the treatment and prevention of Alzheimer's Disease, Alzheimer's disease-related disorder, and down's syndrome neuropathie

Investigations into mechanisms modulating proliferation, differentiation, and apoptosis in cultured liver, adrenal, skin, and bone cells

The intricate modulatory roles played by manifold hormones, growth factors, cytokines, extracellular calcium concentrations, intracellular second messengers, protein kinases, and nuclear poly(ADP-ribose) polymerase in proliferative, differentiative, and apoptotic processes have been the subject of investigations that were carried out by means of in vitro either primary or secondary/tertiary cultures of differentiated epithelial (hepatocytes, keratinocytes, and adrenocytes) and connective tissue cells (osteoblasts and fibroblasts) obtained from man and/or other mammalians. In most cases, an ad hoc model system, in which cells were floated on the top of the growth medium and, hence, could enjoy nearly normal respiratory exchanges, was used. Such a system increased cell viability and the ability of parenchymal epithelial cells to respond to extremely low concentrations of growth factors, hormones, and pharmaco-toxicological agents in a way conceivably very close to their behaviour in vivo

Proteomic analysis of GTP-CH-I-binding proteins in normal adult human astrocytes under both basal and proinflammatory cytokine-activated conditions.

Proteomic analysis of GTP-CH-I-binding proteins in normal adult human astrocytes shows the occurrence of significant changes in the actual protein complexes under both basal and proinflammatory cytokine-activated conditions. Abstract no. 24

Bcl10 crucially nucleates proapoptotic complexes including PDK1, PKCζ, and caspase-3 at the nuclear envelope of etoposide-treated human cervical carcinoma C4-I cells.

Protein kinase  (PK)Cζ signaling at various subcellularlevels affects cell survival, differentiation, growth and/or apoptosis. However, the mechanisms modulating PKCζ activity at the nuclear membrane  (NM) are not yet fully understood. Previously, we demonstrated that PKCζ interacts with the B‑-cell lymphoma  10  (Bcl10) protein at the NM of human cervical carcinoma  (HCC) C4‑-I cells. In the present study, we aimed to further clarify the interactions between PKCζ, Bcl10 and other proteins co-immunoprecipitated from NMs isolated from untreated and etoposide (also known as VP‑-16; 2.0  µg/ml)‑-treated C4‑-I cells using biochemical and proteomics analyses. Aside from the Bcl10 protein, 3‑-phosphoinositide‑-dependent protein kinase‑-1  (PDK1) also co-immunoprecipitated with PKCζ from NMs of C4‑-I cells, indicating the assembly of a heterotrimeric complex, which increased with time in VP‑-16‑-exposed cells, as did the activity of PDK1‑-phosphorylated‑-PKCζ. In turn, PKCζ‑-phosphorylated‑-Bcl10 straddled an enlarged complex which comprised caspase‑-3. Subsequently, activity‑-enhanced caspase‑-3 cleaved and inactivated PKCζ. Finally, the suppressionof Bcl10 using specific siRNA or lentiviral transduction prevented the increase in the PDK1•PKCζ association, the increase in the activity of PKCζ and caspase‑-3, as well as the caspase‑-3‑-mediated PKCζ proteolysis and inactivation from occurring at the NMs of the VP‑-16‑-exposed C4‑-I cells. Our observations provide evidence that Bcl10 acts as a pivotal pro-apoptotic protein which crucially nucleates complexes comprising PDK1, PKCζ and active caspase‑-3 at the NMs of VP‑-16‑-exposed C4‑-I cells. Hence, our data suggest that Bcl10 and PKCζ are potential therapeutic targets in the treatment of HCC

Opposite effects of cell differentiation and apoptosis on Ap3A/Ap4A ratio in human cell cultures.

The biological role of diadenosine oligophosphates (DAOP) remains obscure in spite of numerous attempts to solve this enigma. It is known that Ap3A contrary to Ap4A accumulates in human cultured cells treated with interferons (IFNs) alpha or gamma. Since IFNs are considered as antiproliferative regulators, we assumed that different cell status may be associated with varying intracellular levels of DAOP. Promyelocytic human cell line HL60 induced by phorbol ester (TPA) to differentiate to macrophage-like cells in culture exhibits a profound loss of proliferative potential. Here we have shown a 4-5-fold increase in Ap3A concentration in HL60 cells induced by TPA, similar to the effect of IFN, while the Ap4A concentration remained unchanged. On the contrary, in cells undergoing apoptosis induced by VP16, a topoisomerase II inhibitor, the Ap3A concentration considerably decreased, while the Ap4A concentration increased. These findings combined with earlier results suggest an involvement of the Ap3A/Ap4A ratio in signal transduction pathways controlling the cell status

Changes in nuclear protein kinase C-delta holoenzyme, its catalytic fragments, and activity in polyomavirus transformed pyF111 rat fibroblasts while proliferating and following exposure to apoptogenic topoisomerase-II inhibitors.

Protein kinase C-delta (PKC-delta) appears to be variously involved in proliferation and apoptosis. To compare the changes of this enzyme in these two processes, we have determined the levels and activities of the 79-kDa PKC-delta holoenzyme and its catalytically active 47- and 40-kDa C-terminal fragments in the nuclei of proliferating untreated polyomavirus-transformed pyF111 rat fibroblasts and pyF111 cells treated with the apoptogenic topoisomerase-II inhibitors VP-16 (etoposide), VM-26 (teniposide), and doxorubicin. PyF111 cells were chosen because they hyperexpress PKC-delta and they are hypersusceptible to apoptosis because they do not express the antiapoptotic proteins Bcl-2 and Bcl-XL. The highest PKC-delta activity in cells before they started proliferating or were exposed to one of the inhibitors was in the NM (nuclear envelope-containing) fraction, which contained the holoenzyme and both C-terminal fragments, while only the two fragments were in the nucleoplasmic (NP) fraction where they were tightly associated with chromatin. When the cells began proliferating the amounts of the PKC-delta holoenzyme and the two fragments increased in the NM and the NP fractions and the already high PKC-delta activity either increased or stayed the same in these fractions until the end of the 72-h incubation. And there was no leakage of cytochrome c from the mitochondria into the cytoplasm. VP-16 exposure caused a prompt release of cytochrome c from the mitochondria into the cytosol and at the same time triggered a sharp drop (35% by 3 h and 60% by 6 h) in the PKC-delta activity in the NM fraction without changing the actual amounts of the holoenzyme or its fragments. This prompt inactivation of PKC-delta and its fragments during the first 6 h of exposure to the drug was not due to their dephosphorylation and could not be reversed by phosphatidylserine and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). Between 6 and 24 h the PKC-delta activity in the NM fraction dropped a further 20%, the kinase's activity transiently surged in the NP fraction, and cytoplasmic CPP-32-like (DEVD-specific caspase) activity increased without an increase in the proteolysis of nuclear PKC-delta or PARP. Between 24 and 72 h nuclear CPP-32-like activity increased along with a massive proteolysis of PKC-delta, an accumulation of various PKC-delta fragments, and the cleavage of PARP. But despite this proteolysis, the cells were still able to maintain or even increase the amounts of holoenzyme and 40- and 47-kDa fragments in the NM and NP fractions before dying. VM-26 and doxorubicin caused the same prompt release of cytochrome c from the mitochondria and dramatic drop of NM PKC-delta activity as did VP-16. Thus, high levels of activity of nuclear PKC-delta, particularly PKC-delta in the nuclear membrane, might have a role driving the cell cycle of pyF111 cells. On the other hand, the prompt and sustained large drop in the activity of PKC-delta at this site that precedes the onset of the caspase-mediated proteolysis of the isoform may be involved in starting and driving apoptogenesis in pyF111 fibroblasts exposed to topoisomerase-II inhibitors

Combining immunofluorescence with in situ proximity ligation assay: a novel imaging approach to monitor protein–protein interactions in relation to subcellular localization

The in situ Proximity Ligation Assay (PLA) is suited for visualizing protein–protein interactions and posttranslational protein modifications in both tissue sections and in vitro cell cultures. Accurate identification and quantification of protein–protein interactions are critical for in vitro cell analysis, especially when studying the dynamic involvement of proteins in various processes, including cell proliferation, differentiation, and apoptosis. Here, we monitored the interactions between protein kinase-Cζ (PKCζ) and Bcl10 protein in untreated and etoposide (VP-16)-treated C4-I cells by means of a new combined morphological approach and validated it by taking stock of our previous proteomic and biochemical work (Chiarini et al. in J Proteome Res 11:3996–4012, 2012). We first analyzed the colocalization of PKCζ and Bcl10 proteins through classical immunofluorescent colocalization analysis. On the basis of these results, we developed a novel imaging approach combining immunofluorescence (IF) techniques with in situ PLA to identify the PKCζ·Bcl10 complexes at the level of a specific subcellular compartment, i.e., the nuclear envelope (NE). By this means, we could show that the amount of PKCζ·Bcl10 complexes localized at the NE of C4-I cells during proliferation or after treatment with VP-16 closely corresponded to our previous purely biochemical results. Hence, the present findings demonstrate that the combination of in situ PLA with classical IF detection is a novel powerful analytical tool allowing to morphologically demonstrate new specific protein–protein interactions at level of subcellular organelles, the complexes functions of which can next be clarified through proteomic/ biochemical approaches

Le basi essenziali di Neuroanatomia Clinica e Neurofisiologia

Traduzione di “MANTER’S AND GATZ’S ESSENTIALS OF CLINICAL NEUROANATOMY AND NEUROPHYSIOLOGY” di S. Gilman e S. Winans Newman, 10a ed.F.A. DAVIS CO. PHILADELPHIA, PA, US

BCL10 and PKC-zeta interactions in apoptosis of human cervical carcinoma cells triggered by etoposide

The BCL10/PKC-zeta interactions in the apoptosis of human cervical carcinoma cells triggered by etoposide are preliminarily reporte