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Current literature agrees on the notion that efficient DNA repair favors longevity across evolution. The DNA damage response machinery activates inflammation and type I interferon signaling. Both pathways play an acknowledged role in the pathogenesis of a variety of age-related diseases and are expected to be detrimental for human longevity. Here, we report on the anti-inflammatory molecular make-up of centenarian’s fibroblasts (low levels of IL-6, type 1 interferon beta, and pro-inflammatory microRNAs), which is coupled with low level of DNA damage (measured by comet assay and histone-2AX activation) and preserved telomere length. In the same cells, high levels of the RNAseH2C enzyme subunit and low amounts of RNAseH2 substrates, i.e. cytoplasmic RNA:DNA hybrids are present. Moreover, RNAseH2C locus is hypo-methylated and RNAseH2C knock-down up-regulates IL-6 and type 1 interferon beta in centenarian’s fibroblasts. Interestingly, RNAseH2C locus is hyper-methylated in vitro senescent cells and in tissues from atherosclerotic plaques and breast tumors. Finally, extracellular vesicles from centenarian’s cells up-regulate RNAseH2C expression and dampen the pro-inflammatory phenotype of fibroblasts, myeloid, and cancer cells. These data suggest that centenarians are endowed with restrained DNA damage-induced inflammatory response, that may facilitate their escape from the deleterious effects of age-related chronic inflammation.

Newly generated neurons initiate polarizing signals that specify a single axon and multiple dendrites, a process critical for patterning neuronal circuits in vivo. Here, we report that the pan-neurotrophin receptor p75NTRis a polarity regulator that localizes asymmetrically in differentiating neurons in response to neurotrophins and is required for specification of the future axon. In cultured hippocampal neurons, local exposure to neurotrophins causes early accumulation of p75NTRinto one undifferentiated neurite to specify axon fate. Moreover, knockout or knockdown of p75NTRresults in failure to initiate an axon in newborn neurons upon cell-cycle exit in vitro and in the developing cortex, as well as during adult hippocampal neurogenesis in vivo. Hence, p75NTRgoverns neuronal polarity, determining pattern and assembly of neuronal circuits in adult hippocampus and cortical development.

Immunoglobulin (Ig) isotype diversification by class switch recombination (CSR) is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID). Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca2+-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5′-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

Abstract: Studies in modeled microgravity or during orbital space flights have clearly demonstrated that endothelial cell physiology is strongly affected by the reduction of gravity. Nevertheless, the molecular mechanisms by which endothelial cells may sense gravity force remain unclear. We previously hypothesized that endothelial cell caveolae could be a mechanosensing system involved in hypergravity adaptation of human endothelial cells. In this study, we analyzed the effect on the physiology of human umbilical vein endothelial cell monolayers of short exposure to modeled microgravity (24–48h) obtained by clinorotation. For this purpose, we evaluated the levels of compounds, such as nitric oxide and prostacyclin, involved in vascular tone regulation and synthesized starting from caveolae-related enzymes. Furthermore, we examined posttranslational modifications of Caveolin (Cav)-1 induced by simulated microgravity. The results we collected clearly indicated that short microgravity exposure strongly affected endothelial nitrix oxide synthase activity associated with Cav-1 (Tyr 14) phosphorylation, without modifying the angiogenic response of human umbilical vein endothelial cells. We propose here that one of the early molecular mechanisms responsible for gravity sensing of endothelium involves endothelial cell caveolae and Cav-1 phosphorylation.

Abstract: Studies on the transmission from man to animals of Creutzfeld-Jacob disease (CJD) led Prusiner to identify a proteinaceous infectious particle lacking nucleic acid, which was called prion. The identification of the infectious prion (PrPsc) then led to the discovery of the normal cellular counterpart (PrPc). One of the still enigmatic aspects regarding prion diseases is actually how, where, and when the transformation PrPc/PrPsc is occurring, this being due to the result of a large extent to the fact that so far most studies have been dedicated to the formation and transmission of PrPsc, whereas the understanding of physiologic roles of PrPc are in their infancy. In this review, we hope to identify the most reliable hypotheses for future experiments on PrPc. This is relevant not only for the understanding of PrPc functions but also to unravel the enigmatic nature of PrPc/PrPsc conversion.

In vascular cells, prostacyclin (PGI2) synthase (PGI2s) has been localized in the endoplasmic reticulum of endothelial cells and in the nuclear and plasma membrane of smooth muscle cells. In human umbilical vein endothelial (HUVE) cells, we detected the enzyme in abundant cytoplasmic vesicles apparently originating from the plasma membrane and similar to those stained by gold-albumin, which interacts with a caveolar receptor. This prompted us to try a direct confocal microscopy approach aimed at colocalizing gold-albumin, caveolin-1, and PGI2 synthase. Moreover, the staining of HUVE cells with an anti-BiP7Grp78 antibody (a marker of endoplasmic reticulum) shows a perinuclear localization, sharply separated from PGI2 synthase localization. The results indicate that more than 80% of the enzyme resides in cellular sites costaining with caveolin-1 antibody and gold-albumin. This evidence was confirmed by the demonstration that PGI2 synthase and caveolin-1 coimmunoprecipitate in HUVE cell lysates and that they are associated to detergent-insoluble membrane domains in the same low-density fractions of a sucrose gradient. In addition, depletion of cellular cholesterol by mevalonate and methyl-β-cyclodextrin leads to the shift of PGI2 synthase and caveolin-1 to higher density fractions of the gradient. Biochemical evidence about colocalization was supported by the use of a fusion protein glutathione S-transferase (GST)/caveolin-1, which retained either PGI2s purified from ram seminal vesicles or PGI2s present in HUVE cell lysates. Binding of PGI2s to caveolin “scaffolding domain” and to C-terminal region was deduced by using full-length GST–Cav-1, GST–Cav 61–101, and GST C- and N-terminal fusion proteins. A double approach based on the usage of filipin as a specific caveolae-disrupting agent and antisense oligonucleotides targeting PGI2 synthase mRNA suggests that the production of PGI2 in caveolae is likely to be connected to the regulation of angiogenesis, at least in vitro.

Cystatin B is an anti-protease implicated in myoclonus epilepsy, a degenerative disease of the central nervous system. In vitro,cystatin B interacts with and inhibits proteases of the cathepsin family. Confocal microscopy analysis of the subcellular localization of cystatin B and cathepsin B shows that, in vivo,the two proteins are concentrated in different cell compartments. In fact, cystatin B is found mainly in the nucleus of proliferating cells and both in the nucleus and in the cytoplasm of differentiated cells, while cathepsin B, in either case, is essentially cytoplasmic. However, colocalization of cystatin and cathepsin B is observed in the isolated cell matrix and in the nuclear scaffold of differentiated neuroblastoma cells but not of proliferating cells. This suggests that at least a fraction of cystatin B is bound to the protease in differentiated cells. The electron microscopy analysis of the cell matrix confirms the observation made with confocal microscopy. The cellular activity of cathepsin B was analyzed with a fluorogenic cytochemical assay. A fluorescent signal is observed in the cytoplasm of proliferating cells but is undetectable in the cytoplasm of differentiated cells, suggesting that cathepsin B is active mainly during the cell cycle. This result is consistent with the separate compartimentalization of cystatin B and cathepsin B that we have observed in growing cells.

Knock-out of the gene coding for caveolin-1, the main organizer of caveolae, has not yet been performed. We devised a strategy to knock-down caveolin-1 gene expression using antisense oligodeoxynucleotides (ODNs). Seven ODNs, covering different regions of caveolin-1 mRNA, were screened by Western blot analysis of caveolin-1 levels. The most active and specific was found to reduce caveolin-1 protein levels by 70% at 1 μM concentration and its action, as demonstrated by a marked reduction (about 50%) in caveolin-1 mRNA levels, was due to a true antisense mechanism. In HUVEC treated with the active ODN, caveolae were undetectable by confocal and electron microscopy, while their number was not affected when cells were treated with a scrambled ODN. Using the fibrin gel 3 D angiogenesis test we established that the active (but not the scrambled) ODN strongly suppressed capillary-like tube formation. Moreover, an antisense tailored against chicken caveolin-1 mRNA, when tested using the chorio-allantoic membrane technique, dramatically reduced vessel formation at doses (10–20 μg) under which control ODNs were ineffective and devoid of toxicity. Thus, it is likely that caveolin-1 down regulation, followed by caveolae disruption, impairs angiogenesis in vitro and in vivo.

An increasing body of evidence shows that many of the key inositol lipids and enzymes responsible for their metabolism reside in nuclei. Moreover, the association of the nuclear phosphoinositide cycle with progression through the cell cycle and commitment toward differentiation has built a wider picture of the implications of phosphoinositides in the control of nuclear functions. This article reviews a central aspect of inositide nuclear signaling, i.e., the spatial organization of the signaling system within the nucleus in relationship to the nuclear organization in functional domains. Most of the evidence obtained with a variety of confocal and electron microscopy immunocytochemical techniques indicates that the phosphoinositides, the enzymes required for their synthesis and hydrolysis, and the targets of the lipid second messengers are localized at ribonucleoprotein structures involved in the transcript processing in the interchromatin domains. These findings demonstrate that nuclear inositol lipids exist in a nonmembranous form, linked to structural nuclear proteins of the inner nuclear matrix. They also suggest that the inositol signaling in the nucleus is completely independent of that at the cell surface and that it probably preceded in evolution the systems that are present at the cytoskeletal and cell membrane level. J. Cell. Physiol. 181:203–217, 1999. © 1999 Wiley-Liss, Inc.

Efflux of chemotherapy agents by P‐glycoprotein at the plasma membrane is thought to be a major cause of cancer multidrug‐resistance (MDR). However, the mechanism underlying the cellular accumulation and distribution of cytotoxic drugs is still poorly defined. We have recently found that P‐glycoprotein is expressed also in the nucleus of MDR cell lines selected in doxorubicin (DXR), suggesting the possible involvement of this protein in the direct extrusion of the drug from the nucleus of resistant cells. In this study, we analyzed the subcellular localization of P‐glycoprotein in a series of U‐2 OS osteosarcoma cell clones transfected with MDR1gene in order to verify whether the nucleus is a constant site for the localization and functional activity of P‐glycoprotein, and in which way some aspects of cell morphology related to MDR depend on the subcellular P‐glycoprotein localization rather than on the exposure to the selective drug. Our results indicate that to achieve a subcellular drug distribution prevailing in the cytoplasm but not in the nucleus, a significant increase in the expression of P‐glycoprotein at the different cellular compartments, including the plasma membrane, the cytoplasm, and the nucleus, is needed, although the in vitrodrug resistance appears to be mainly dependent on the expression of P‐glycoprotein at the cell surface. With regard to the morphological characteristics of MDR cells involving the cell surface and the chromatin arrangement, the influence of DXR appears to be prevalent, although P‐glycoprotein overexpression cannot be excluded.

Abstract:  The intracellular localization of phosphatidylinositol 3-kinase (PI 3-kinase) has been analyzed by western blotting, confocal, and electron microscopy immunocytochemistry in human osteosarcoma Saos-2 cells. By western blotting, the enzyme appears to be present in both the cytoplasmic and nuclear subfractions. By confocal microscope immunocytochemistry, the cytoplasmic fluorescence is localized in the perinuclear region and on a network of filaments, while a diffused signal is present in the nucleus, except for the nucleolar areas. Ultrastructural analyses on whole cells and on in situ matrix preparations reveal that nuclear PI 3-kinase is localized in interchromatin domains, in stable association with inner nuclear matrix components, while the enzyme diffused in the cytosol is partly associated with the cytoskeletal filaments. Quantitative evaluations indicate that, in a multidrug-resistant variant obtained by continuous exposure of Saos-2 cells to doxorubicin, the amount of nuclear and cytoplasmic PI 3-kinase is significantly lower than in the sensitive parental cell line. The nuclear localization of PI 3-kinase and its variation in multidrug-resistant cells, characterized by a reduced mitotic index, are consistent with the data on the existence of a nuclear inositol lipid cycle, which could also utilize 3-phosphorylated inositides to modulate signal transduction for the control of some key functional activities.

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactions

AbstractMigrating primordial germ cells of Rana dalmatinaembryos were labelled by Dolichos biflorusagglutinin. Confocal microscopy analysis demonstrates the nuclear localisation of the labelling. The glycoconjugate is expressed by primordial germ cells throughout the entire migration process, but it is no longer detectable in late intragonadal primordial germ cells. These data suggest that the a‐N‐acetylgalactosamine‐containing glycoconju‐gates in the nucleus of primordial germ cells may play a functional role in primordial germ cells protection against differentiation during the migration process. The temporal correlation between disappearance of nuclear Dolichos biflorusagglutinin positivity and colonisation of the gonadal ridges, may be explained by a change in the functional status of primordial germ cells nuclear glycoconjugates in relation with the onset of primordial germ cells differentiation.

Abstract:  DNA sequences digested by HaeIII and reconstructed by in situ nick translation employing digoxigenin-labelled nucleotides are usually revealed either by horseradish peroxidase or FITC fluorescence. To obtain a significant improvement in terms of resolution, sensitivity and specificity, colloidal gold has been used instead of FITC (as the reporter molecule) to reveal the labelled DNA. Colloidal gold and propidium iodide were visualised by employing the reflectance mode and the 488-nm laser line of a confocal laser scanning microscope. In chromosomes, the fluorescent reaction pattern showed diffuse areas of labelling in which it was impossible to identify any specific kind of banding along the arms. In some chromosomes and, in particular, 1 and 9, a C-negative banding due to the negativity of the centromeric areas was seen. A more accurate localisation on chromosomes, including telomeric regions, often organised in spot pairs that resembled an R-like banding, was detected using 1-nm colloidal gold. A fine labelling was also demonstrated in nuclei, especially at their peripheral heterochromatin. The non-fading properties of colloidal gold combined with visualisation by reflectance confocal laser scanning microscopy demonstrated the possibility of obtaining a higher spatial resolution than when using conventional fluorophores or higher laser wavelength. This improved way to study the localization of HaeIII digestion sites in single chromosomes and in interphase nuclei made the reaction a valuable tool for the detection of antigens or of specific DNA sequences in biological preparations.

Biogenesis of prostanoids is under the control of some polypeptide growth factors. Cytosolic phospholipase A₂, a form specific for arachidonic acid containing phospholipids, is activated by a translocation mechanism regulated by growth factors, while prostaglandin H synthase isoforms are induced de novo in several cell types. No information is available as far as PGI₂ synthase is concerned. Human umbilical vein endothelial cells were cultured under conditions favoring proliferation or differentiation or capillary-like network formation in the presence of collagen gels. Basic fibroblast growth factor (bFGF 0.5-4 ng/ml) was used as a mitogen, interleukin-1α (IL-1α 10-60 UI/ml) as a differentiating agent, and prostacyclin (PGI₂) biosynthesis was evaluated. Under the first condition, basal PGI₂ production was unaffected while, in the presence of IL-1α, a marked stimulation of PGI₂ synthesis was observed. It is known that IL-1α is a potent inducer of PGH synthase, while it is not known whether PGI₂ synthase is also induced. Two lines of evidence indicate that PGI₂ synthase is a constitutively expressed not inducible enzyme: (a) proliferating nonproducing cells when added with PGH₂ produce an amount of PGI₂ not different from the amount produced by cells stimulated with IL-1α; (b) under this condition PGI₂ synthase was immunodetectable either by immunofluorescence detected by confocal microscopy or by ELISA and, on microsomes isolated from endothelial cells, by Western blotting. It is concluded that the limiting step in the conversion arachidonate-PGI₂ is represented solely by the level of PGH synthase. These results strongly suggest, but do not prove, the constitutive nature of the enzyme. The final demonstration requires the availability of a probe to detect mRNA level, a trial we are carrying out at the moment. Copyright 1995, 1999 Academic Press

Tiazofurin, an anticancer drug which inhibits IMP dehydrogenase, decreases cellular GTP concentration, induces differentiation and down-regulates ras and myc oncogene expression, caused apoptosis of K562 cells in a time- and dose-dependent fashion. Apoptotic cells were detected by (1) flow cytometry, (2) electron microscopy, and (3) fluorescence in situ nick translation and confocal microscopy, while the DNA ladder was not detectable. The induced apoptosis was abrogated by guanosine which replenishes GTP pools through the guanosine salvage pathways, while it was enhanced by hypoxanthine, a competitive inhibitor of GPRT. The tiazofurin-mediated apoptosis may therefore be linked with the decrease of GTP and the consequent impairment of specific signal transduction pathways. Tiazofurin induced apoptosis also in lymphoblastic MOLT-4 cells, suggesting that this action is not confined to cells of the myeloid lineage, where the differentiating effects of the drug are more pronounced. Cytometry 30:61–66, 1997. © 1997 Wiley-Liss, Inc.

Abstract:  The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.

Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-a,-e,-? and-?. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the a- and e-isoforms. The 80-kDa native form of PKC-a almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C-e appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant inthe Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions. Moreover, the expression of the a- and e-isoforms could be regulated by sexual hormones, as suggested by the differential abundance of these two proteins in the gland of pregnant mouse compared with normal female mouse.

Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II α and β isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al.,1992, Exp. Cell Res.200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II α are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II β is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II α present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II α content is only slightly decreased. On the other hand, the great majority of topoisomerase II β is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topiosomerase types.