Poly(ADP-ribose) synthesis: a useful parameter for identifying apoptotic cells

Cell death by apoptosis was analysed in HeLa cells either treated with the antitumoral drug bleomycin or depleted of growth factors by long-term culture without medium change. The interference of apoptosis with normal cell cycle progression was followed by flow cytometry in cells stained with propidium iodide and with antibody to S-phase-related PCNA protein. Bleomycin-treated cells showed a net accumulation in G₂/M phase paralleled by the appearance of material with a subdiploid DNA content. Cells with a subdiploid DNA content were also present in growth factor-depleted cultures and were shown to derive from all the cell cycle phases. To identify apoptotic features in HeLa cell cultures, we applied a recently developed assay based on the simultaneous analysis in the single cell of three parameters, namely chromatin condensation, DNA degradation and poly(ADP-ribose) synthesis. Apoptotic cells were visualized by sequential reactions: Hoechst staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and immunoreaction with anti-poly(ADP-ribose) monoclonal antibody. Positive reactions were obtained for cells at different stages of the apoptotic programme showing condensed nuclei, fragmented chromatin and apoptotic bodies This revised version was published online in November 2006 with corrections to the Cover Date.     

Expansive growth of the nuclear envelope and formation of mitochondria in ganglionic neuroblasts

Summary Many neuroblasts undergo differentiation in the lateroventral region of spinal ganglia in chick embryos at the 4th to 5th incubation day. In these neuroblasts a striking increase in the chondriome has been recorded, and the following structures have been observed. a) Whorls of membranes which are continuous with the outer nuclear membrane: ribosomelike granules lie in the intramembranous spaces of these whorls, b) Membranous whorls with a peripheral portion, crescent-shaped in the sections, containing a minute granular material, c) Membranous whorls which are continuous with the membranes of the envelope, and of the cristae of large, irregular mitochondria. d) Formations which are transitional in structure between the ones mentioned above.No significant differences were recorded of the thickness of the outer nuclear membrane, of the whorls' membranes, and of the mitochondrial membranes. Beside, the mean values of the total area of the membranes of each whorl of the type listed under a), and of the total area of the membranes of large mitochondria, such as listed under c), are of the same order of magnitude.

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Zusammenfassung Während des 4. und 5. Bruttages differenzieren sich im lateroventralen Abschnitt der Spinalganglien des Hühnerembryos viele Neuroblasten. In diesen Neuroblasten nimmt das Chondriom stark zu und man findet gleichzeitig folgende Strukturen: a) Rollen, aus Membranen gebildet, die einen komplizierten, von Fall zu Fall verschiedenen Verlauf aufweisen. Die Rollen setzen sich in die äußere Nuclearmembran fort. Zwischen ihren Membranen liegen elektronenoptisch dichte, den Ribosomen ähnelnde Granula. b) Membranrollen, deren peripherer halbmondförmiger Abschnitt feinkörniges Material enthält. c) Rollen, deren Membranen sich in die Außenmembran und die Cristae umfangreicher, unregelmäßig geformten Mitochondrien fortsetzen. d) Übergangsformen zwischen denen unter b) und c) beschrieben.Die äußere Nuclearmembran, die Membranen der beschriebenen Rollen und der Mitochondrien weisen die gleiche Dicke auf. Die Membranen, welche eine der unter a) geschilderten Rollen bilden und jene, die sich in einem der umfangreichen Mitochondrien befinden (c), haben Oberflächen von gleicher Größenordnung.Unter den verschiedenen Hypothesen zur Interpretation der Befunde verdient folgende den Vorzug, da sie eine klare Deutung aller gewonnenen Befunde erlaubt: die äußere Nuclearmembran dehnt sich aus und führt auf diese Weise zur Entstehung einer Membranrolle. In dieser Rolle bildet sich sodann ein körniges Material, den Vorläufer der MitochondrienMatrix. Die Menge des körnigen Materials nimmt zu, gleichzeitig wickeln sich nach und nach die Membranenrollen ab und bilden die Hülle und die Cristae eines umfangreichen Mitochondrions. Letzteres teilt sich endlich in mehrere kleinere Mitochondrien auf. Während der Neuroblastendifferenzierung der Spinalganglien des Hühnerembryos geht also die Bildung von Mitochondrien von der Nuclearhülle aus.

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Research supported by a C.N. R. grant.

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I wish to thank Prof. R. Amprino and Prof. J. D. Robertson for much useful discussion and criticism, Dr. phis. S. de Petris for the help given in the statistical evaluation of the data, Mrs. G. Fiori for technical assistance, and Miss A. Bertolasi for drawing Fig. 17.     

L'Organizzazione strutturale delle lamine gliali periventeicolari dell'uomo in condizioni normali e patologiche

Summary The Autor has studied in man the structure of the periventricular glial membranes (corneal laminae) of lateral ventricles of normal and hydrocephalic brains. In normal, these membranes are constituted of three layers: ependyma, sub-ependymal fibrillar layer and vascular layer, whose thickness varies according to the region.In advanced hydrocephalus, the corneal laminae become notably thicker building up a plurilaminar structure, while many gliocytes undergo a clear hypertrophy accompanied with an increase of the intracellular proteic substance. These findings confirm the mechanic significance of the glial structures and the possibilities of structural adaptation of fibrous gliocytes to modified mechanical conditions.The structural peculiarities of corneal cytomorphosis of fibrous gliocytes allow to establish an interesting comparison with the behavior of the epidermal cells.     

Osservazioni morfologiche e considerazioni funzionali sulla muscolaris mucosae dello stomaco dell'uomo

Riassunto La muscolaris mucosae passando dall'esofago allo stomaco presenta una suddivisione e poi uno sfioccamento dei fasci muscolari, che si risolvono in singole file di cellule: queste vanno a costituire una lamina continua espansa in superficie, i cui elementi sono diretti in ogni senso. Da questa lamina, che sulla faccia interna presenta un gran numero di concavità, si staccano gruppi di cellule che circondano a spirale i tubuli ghiandolari, dirigendosi verso la superficie libera della mucosa: la mucosa risulta quindi compenetrata da un dispositivo muscolare, la cui contrazione determina la spremitura delle ghiandole, delle vene e dei linfatici.Nella parte pilorica, dove la muscolaris mucosae è più robusta, in relazione alla riduzione di calibro del viscere le cellule muscolari tendono a riunirsi nuovamente in fascetti diretti in ogni senso. In corrispondenza del piloro le cellule muscolari si riuniscono in fasci a forma di robusti nastri disposti in strati sovrapposti ed assumono nuovamente, come nel cardia, una direzione longitudinale. Contraendosi, esse determinano la formazione di una piega anulare della mucosa, che può ridurre od occludere il lume, con l'effetto di rinforzare o completare o perfezionare l'azione dello sfintere.     

[3H] spiroperidol binding to a putative dopaminergic receptor in rat pituitary gland

A class of high affinity DA receptor sites has been identified with [³H] SPIR in the anterior and pisterior lobes of rat pituitary gland. Competitive studies with DA receptor antagonists and agonists clearly demonstrated that [³H] SPIR stereospecifically labels a true dopaminergic receptor in both lobes. The physiological significance of these receptors, although still unclear, may be that of controlling the secretion of pituitary hormones and peptides.     

Retinoic acid, GABA-ergic, and TGF-beta signaling systems are involved in human cleft palate fibroblast phenotype

During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-beta binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA--which, at pharmacologic doses, induces cleft palate in newborns of many species--were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-beta signaling systems could be involved in human cleft palate fibroblast phenotype.     

Mesoporous silica chips for selective enrichment and stabilization of low molecular weight proteome

The advanced properties of mesoporous silica have been demonstrated in applications, which include chemical sensing, filtration, catalysis, drug delivery and selective biomolecular uptake. These properties depend on the architectural, physical and chemical properties of the material, which in turn are determined by the processing parameters in evaporation‐induced self‐assembly. In this study, we introduce a combinatorial approach for the removal of the high molecular weight proteins and for the specific isolation and enrichment of low molecular weight species. This approach is based on mesoporous silica chips able to fractionate, selectively harvest and protect from enzymatic degradation, peptides and proteins present in complex human biological fluids. We present the characterization of the harvesting properties of a wide range of mesoporous chips using a library of peptides and proteins standard and their selectivity on the recovery of serum peptidome. Using MALDI‐TOF‐MS, we established the correlation between the harvesting specificity and the physicochemical properties of mesoporous silica surfaces. The introduction of this mesoporous material with fine controlled properties will provide a powerful platform for proteomics application offering a rapid and efficient methodology for low molecular weight biomarker discovery.