Biomedical subjects

R Bareggi

Publications and source records attributed to R Bareggi.

At least 19 recordsLinked to original sources

PubMed1997

Association of nuclear matrix proteins with granular and threaded nuclear bodies in cell lines undergoing apoptosis.

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyer et al., Exp. Cell Res. 221,27-40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-alpha, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequences in vitro, thus mediating the formation of looped DNA structures in vivo. Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.

Antigens, Nuclear↗

PubMed1996

On the assessment of the growth patterns in human fetal limbs: longitudinal measurements and allometric analysis.

The total length (TL) and length of the ossified part (OL) of some long bones of the upper (humerus, ulna, radius) and lower limb (femur, tibia, fibula) were evaluated in 58 aborted human fetuses (crown-rump length, CRL, between 38 and 116 mm, developmental age from 8 to 14 weeks). The specimens, without any detectable malformation, were cleared and double-stained with alcian blue and alizarin red S to obtain a differential detection of the ossified part within the comprehensive outline between the cartilaginous epiphyses. The correlation between the values of TL and OL and those of CRL emphasized that the systematic OL measurement in limb long bones correlated better than TL with development age, since OL increased faster than TL. TL and OL also correlated with the CRL by bivariate allometry (ln y = ln a + b ln x) and the data obtained showed that they grew with positive allometry. The comparison between the cumulative values of the bones examined in each limb showed that both TL and OL grew relatively faster in the lower limb than the upper; the greatest growth rate was found for OL in the lower limb. These results many provide a tool for a comprehensive assessment of long bone growth patterns and may be useful in determining fetal growth even in incomplete specimens, in which one or some long bones can still be measured.

Embryonic and Fetal Development↗

PubMed1996

Stimulation of nuclear polyphosphoinositide synthesis by GTP-gamma-S: a potential regulatory role for nuclear GTP-binding proteins.

The nonhydrolyzable GTP analogue GTP-gamma-S was capable of stimulating in vitro phosphorylation of polyphosphoinositides in isolated nuclei prepared from mouse erythroleukemia cells. On the contrary, GDP-beta-S was ineffective. The stimulation was not detectable when nuclei were prepared from erythroleukemia cells induced to differentiate by exposure to dimethyl sulfoxide. Both nuclear phosphomonoesterase and phospholipase C activities were not influenced by GTP-gamma-S. Our results point to the likelihood that nuclear phosphoinositide kinases might be regulated by a GTP-binding protein.

Animals↗

PubMed1996

Expression and immunohistochemical localization of eight phospholipase C isoforms in adult male mouse cerebellar cortex.

By means of specific polyclonal or monoclonal antibodies we have investigated the expression and the localization of phospholipase C isoforms in the adult mice cerebellar cortex. Western-blot analysis revealed that mouse cerebellum expressed eight phospholipase C isozymes: -beta 1, -beta 2, -beta 3, -beta 4, -gamma 1, -gamma 2, -delta 1, -delta 2. Immunohistochemical analysis carried out on cryosections showed a distinct pattern of expression for each of the isoforms. Purkinje cells had high levels of -beta 1, -beta 3, -gamma 2 and -delta 2 isotypes. The -gamma 2 isozyme was the only one that was identified also in the dendrites of Purkinje cells. In the molecular layer we detected mostly -beta 1 and -gamma 1 isozymes whereas in the granular layer -gamma 1 and -gamma 2 isoforms prodominated. These results indicate a heterogeneity of the phospholipase C isoforms expressed in the layers of mouse cerebellar cortex conceivably due to the fact that these enzymes are coupled to different receptors and perform selective tasks in regulating cell signalling events taking place in the cerebellar cortex of mice.

Amino Acid Sequence↗

PubMed1996

Selective distribution of multiple protein kinase C isoforms in mouse cerebellar cortex.

An immunohistochemical study concerning the distribution of protein kinase C isoforms, a lipid-regulated serine/threonine kinase essential for signal transduction, was performed in mice cerebellar cortex, with particular emphasis on the localization of -iota and -lambda isozymes. By the means of immunoblotting analyses we detected the presence of 11 PKC subspecies in whole cerebellar extracts. Immunoreactivity on cryostat sections revealed, using polyclonal and monoclonal antibodies, that a few isoforms were widely but discretely distributed in all three cortical layers (molecular, granular and Purkinje cells) whereas other isozymes were present in a limited neuronal compartment. Overall, the distribution of several isoforms was in agreement with data obtained by other authors using rat cerebellum. As far as -iota and -lambda isozymes were concerned, we found them abundantly expressed in endothelial cells. Moreover, protein kinase C-lambda was also present in the body of Purkinje cell, conceivably associated with a 200-kDa neurofilament component. In all, these results hint at the possibility that in the cerebellar cortex at least some protein kinase C isoforms are involved in functions other than signal transduction at the synaptic level.

Animals↗

PubMed1995

Diffuse vesicular distribution of Rab3D in the polarized neuroendocrine cell line AtT-20.

The neuroendocrine cell line AtT-20 has two types of storage vesicles: dense core granules and synaptic vesicles, both sequestered at the tip of the processes. Here we show that Rab3D protein, which is abundant in fat cells, is also expressed in AtT-20 cells. Differently from Rab3A, which is localized in secretory vesicles accumulated at the tips, Rab3D has a diffuse vesicular distribution in the cytoplasm of the cell body, the processes and the tips. In AtT-20 cells, Rab3D may define a regulated secretory pathway which functions independently from cell polarity.

Amino Acid Sequence↗

PubMed1995

Behavior of nuclear matrix proteins during camptothecin-induced apoptosis in HL-60 human leukemia cells.

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

Antigens, Nuclear↗

PubMed1995

Distribution of the extended family of protein kinase C isoenzymes in fetal organs of mice: an immunohistochemical study.

Using isoenzyme-specific antisera, we have studied the distribution of protein kinase C isoforms in fetal mouse organs at the developmental age of 17 days. Two different sets of antibodies, produced by different manufacturers, were employed in this study. The specificity of the antisera was tested by immunoblotting experiments using whole fetal mouse extracts. Immunohistochemistry was carried out by means of an alkaline phosphatase-conjugated secondary antibody. Analysis of fetal mouse longitudinal cryostat sections stained with the antibodies demonstrated a distinct distribution of protein kinase C isoforms in the tissues. Protein kinase C-alpha and C-beta I were present in all tissues examined, whereas the C-beta II isoform was absent in the lung and the liver. Protein kinase C-gamma was identified in brain, spinal ganglia, and adrenal gland. The C-epsilon isoenzyme was abundantly expressed in spinal ganglia and in the smooth muscle cells of the bronchial wall. Antisera to C-zeta and C-eta isoforms heavily stained liver, kidney, and spinal ganglia, whereas the C-theta isozyme was mainly detected in brain, stomach and kidney. Thus, protein kinase C-alpha, C-beta I, C-beta II, C-zeta, C-eta and C-theta were the isoforms present in many of the organs investigated. The two sets of antibodies gave slightly different results that might be ascribed to the different epitopes recognized by the antisera. One set of antisera was employed to investigate the distribution of the isoforms in selected organs from an earlier developmental age (15 days) and from adult animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Protein kinase C isoenzymes in mouse harderian gland. Differential expression of the alpha- and epsilon-isoforms during pregnancy. Protein kinase C-OC.

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-alpha, -epsilon, -zeta and -eta. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the alpha- and epsilon-isoforms. The 80-kDa native from of PKC-alpha 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-epsilon 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 in the 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Mandibular growth rates in human fetal development.

A morphometric analysis of changing proportions in the developing mandible was undertaken in 18 human embryos and fetuses of both sexes (developmental age from 8 to 14 weeks, crown-rump length, CRL, from 34 to 110 mm), previously cleared and stained with a specific method for bone (alizarin red S). Reference points were located on the mandible, i.e. condylar process (Pcl), coronoid process (Pco), gnathion (GN), gonion (GO), superior symphyseal point (SSP), for measuring linear dimensions, i.e. Pcl-GN, Pcl-Pco, Pco-GN, GO-GN, SSP-GN. The gonial (Pcl-GO-GN) and the (Pcl-GN-Pcl) angles were also measured. All linear dimensions were correlated with the CRL by bivariate allometry (1n y = 1n a+b 1n x): they all grew with positive allometry, except GO-GN with isometry. The mandibular ramus grew relatively faster than the body, both in length and height, and the greatest growth rate was found for ramus height. The relation between mandibular shape and the craniofacial structures was investigated using scale drawings obtained from photographs of fetal skulls in lateral view. In the youngest fetuses the mandible was prognathic, then became retrognathic. During the period investigated the zygomatic process and squama of the temporal bone were in a lower and more inclined position in relation to the transverse plane passing through the zygomatic arch than in the newborn and adult. This study identifies parameters fitting changing trends in height, length and shape of the human mandible during the prenatal period (8-14 weeks); moreover, it emphasizes that the mandibular growth patterns differ significantly from those of successive development periods.

Adult↗

PubMed1995

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins.

Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at 0 degrees C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37 degrees C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37 degrees C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37 degrees C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37 degrees C in vitro, unlike to that what previous reports have indicated.

Animals↗

PubMed1995

A simple and rapid staining technique for plastic embedded cartilage and bone.

In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semithin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na2 CO3, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.

Animals↗

PubMed1994

Catalytic properties of DNA polymerase alpha activity associated with the heart-stabilized nuclear matrix prepared from HeLa S3 cells.

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase alpha associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37 degrees C prior to subfractionation, as has been reported previously for DNA polymerase alpha bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase alpha activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase alpha activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase alpha activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2 M NaCl or 0.25 M (NH4)2SO4 and led us to consider that a 37 degree incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase alpha which is unlikely to be involved in DNA replication in vivo.

Adenosine Triphosphate↗

PubMed1994

The effect of in vitro heating on the distribution of nuclear matrix polypeptides in HeLa cells.

The in situ nuclear matrix was obtained from HeLa cells. After permeabilization with nonionic detergent, the resulting structures were incubated for 1 h at 37 degrees C to determine whether or not such an incubation might result in the redistribution of nuclear polypeptides which resisted extraction with buffers of high-ionic strength (1.6 M NaCl or 0.25 M (NH4)2SO4 as well as DNase I digestion. Using indirect immunofluorescence experiments and monoclonal antibodies we show that heating to 37 degrees C changes the distribution of a 160 kDa protein previously shown to be a component of the inner matrix network. On the other hand, a 125 kDa polypeptide was not affected at all by the incubation. Our results clearly indicate that the inclusion of a 37 degrees C incubation (for example during digestion with DNase I) in the protocol to obtain the in situ nuclear matrix can result in the formation of in vitro artifacts.

Blotting, Western↗

PubMed1994

6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei.

Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37 degrees C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37 degrees C in comparison with those maintained at 0 degrees C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37 degrees C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37 degrees C.

Animals↗

PubMed1994

An immunohistochemical study of protein kinase C distribution in fetal mouse vertebral column.

Using polyclonal antibodies we have studied the distribution of protein kinase C in fetal mouse low thoracic vertebrae. By means of a pan protein kinase C antiserum recognizing the catalytic domain of the enzyme, we show that protein kinase C is markedly expressed in chondrocytes before birth. The enzyme seems to be very abundant in the more mature cells that are close to ossification centres as well as the periphery of the intervertebral disc, although it can also be detected in chondrocytes. In order to establish which protein kinase C isoenzyme(s) the chondrocytes produce, we employed polyclonal isoenzyme-specific antisera developed against three calcium-dependent isoforms (alpha, beta, gamma) and three calcium-independent isoforms (delta, epsilon, zeta). Secondary antibody conjugated to alkaline phosphatase revealed that chondrocytes markedly express the beta-isoform. Cells were also weakly stained by the anti-epsilon serum. The immunostaining was completely abolished by pre-incubating primary antibodies with the peptide antigens to which they were raised. These results suggest that protein kinase C (and particularly the beta isoform) could play an important role in mouse fetal chondrogenesis of the vertebral column.

Alkaline Phosphatase↗

PubMed1994

The growth of long bones in human embryological and fetal upper limbs and its relationship to other developmental patterns.

Measurements were made of the long bones of the upper limbs (humerus, ulna, radius) of 58 aborted embryos and fetuses, developmental age from 8 to 14 weeks, crown-rump length (CRL) between 38 and 116 mm. The specimens were cleared and double-stained, using alcian blue and alizarin red S for a differential detection of cartilage and bone. The values of both the total length (TL) and the ossified part (OL) of each long bone were related to the fetal developmental age previously estimated by freshly measured CRL. The relationship to another developmental pattern, i.e. the number of ossified centres in the vertebral column, suggested that the OL values could be much more significant than TL for the assessment of fetal growth.

Embryo, Mammalian↗

PubMed1994

On the presence of a secondary cartilage in the mental symphyseal region of human embryos and fetuses.

The presence of a secondary cartilage in the mental symphyseal region was examined in this study. A double-staining method with alcian blue and alizarin red S was performed on both whole human embryos and fetuses (developmental age between 8 and 17 weeks, crown-rump length, CRL, between 37 and 124 mm) and their disjointed mandibles. Histological and histochemical techniques were applied to transverse serial sections of whole disjointed fetal heads. The ossification process observed in the mental symphysis is quite different from that of the mandibular body, whose membranous ossification is induced by the contiguous Meckel's cartilage. No evidence of any fusion of Meckel's cartilage with the symphyseal cartilage, that lies within the symphyseal space, was detected. On the basis of these findings, we suggested that the mental secondary cartilage is able to change into bone according to an endochondral ossification process. Moreover, the role of mechanical causes in the development of the mental symphysis was hypothesized.

Cadaver↗