Molecular targeted therapies have changed the landscape of cancer research. Agonistic monoclonal ... more Molecular targeted therapies have changed the landscape of cancer research. Agonistic monoclonal antibodies (MoAbs) targeting TRAIL-death receptors (TRAIL-Rs) have been developed and currently used in clinical trials. Binding of such antibodies to TRAIL-R1 and TRAIL-R2 results in death inducing signalling complex (DISC) formation and induction of apoptosis, which represents a natural mechanism of cell growth control and an ideal target for drug development. These novel fully humanized compounds have been associated with conventional chemotherapy in the treatment of advanced solid malignancies, including different types of lymphoma. Here we outline the rationale and potential of a new molecular-based strategy combining agonistic anti-TRAIL-death receptor monoclonal antibodies plus the pioneer of the new biological frontiers of cancer therapy: rituximab.
Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia
A triploid fetus (karyotype 69, XXX) with crown-rump length (CRL) 94 mm, presenting micro- and re... more A triploid fetus (karyotype 69, XXX) with crown-rump length (CRL) 94 mm, presenting micro- and retrognathia, low-set ears and crooked feet, was cleared and double-stained with alizarin red S and alcian blue for detecting the ossification patterns in the vertebral column, ribs, ischium, limbs, and face. Longitudinal measurements of some long bones in the upper (humerus, ulna, radius) and lower (femur, tibia, fibula) limb were taken. The values of both the total length (TL) and the ossified part (OL) of each long bone, as well as the OL/TL per cent ratio were considered. 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. Since the aim of this work was to assess the influence of triploidy 69, XXX the skeletal development and growth patterns, all values obtained in the examined specimen were related with those relative to a group of fetuses, witho...
Quintessence international (Berlin, Germany : 1985)
This short-term (72- to 96-hour) in vitro study on fibroblasts evaluated the biocompatibility of ... more This short-term (72- to 96-hour) in vitro study on fibroblasts evaluated the biocompatibility of 3 single-phase dental alloys by determining cellular proliferation rates and the expression of a glycoprotein, fibronectin, which is involved in cellular adhesion processes. Flow 2002 fibroblasts were cultured together with 3 single-phase dental alloys of different composition. Proliferation rates were determined by 5-bromodeoxyuridine incorporation. Fibronectin expression was determined by indirect immunofluorescence. At 72 hours, cells cultured with the alloy containing the lowest amount of noble elements (gold, platinum, and palladium) and the highest amount of silver exhibited significantly less proliferation than did controls. At 96 hours, only cultures with the alloy containing the greatest amount of noble elements behaved in a way similar to controls. Fibronectin organization in fibrils and in focal adhesions was correlated to higher cellular proliferation rates. Fibronectin organ...
Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia
In our previous work we have investigated the expression of the serine-threonine kinase protein k... more In our previous work we have investigated the expression of the serine-threonine kinase protein kinase C (PKC) in the vertebral column of mouse foetuses. In the present work we would verify the expression of four PKC-isoenzymes (alpha, delta, epsilon, zeta) in two distinct phases of the chondrogenesis and the endochondral osteogenesis in vitro. We performed primary cultures of chondrocytes collected from tibiae of 6-day old chick embryos. This cells were cultured for 20 days and than collected on coverslips (stage 1 culture). Other cells of the stage 1 were undergone further differentiation towards the phenotype of osteoblast-like cells (stage 2 culture), in accord to the protocol of Descalzi Cancedda et al. (1992). In stage 1 culture, PKC-epsilon was the most expressed isoform, whereas PKC-alpha exhibited the least intense positivity. In stage 2 culture, PKC-alpha was the most expressed isoform, whereas a marked decrease of PKC-epsilon expression was detected compared to stage 1. N...
Bollettino della Società italiana di biologia sperimentale
Recent studies have shown that protein kinase C (PKC) plays a pivotal role in many cellular funct... more Recent studies have shown that protein kinase C (PKC) plays a pivotal role in many cellular functions, i.e. cell proliferation and differentiation, exocytosis, ion-exchange regulation, hormone and neurotransmitter release, programmed cell death. Up to now the tissue and organ distribution of PKC isoenzymes have been studied in various mammalian adults and it has been suggested that each of them could play a specific role in the regulation of various cellular functions. However, few works have been performed on the expression of the enzyme in actively proliferating and differentiating tissues, i.e. during embryonal and fetal developmental stages. In the present study we have performed an immunohistochemical analysis by using polyclonal antibodies in order to verify the distribution of nine PKC isoenzymes in the vertebral column of human fetuses in a key period (8th developmental week), when the most relevant chondrogenetic and osteogenetic processes occur. The detection of the variou...
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) up-regulated the expression of co... more Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) up-regulated the expression of constitutive cyclooxygenase (COX)-1 protein in HL-60 cells without affecting COX-2. The TRAIL-mediated COX-1 up-regulation was accompanied by a significant increase of the PGE(2) synthesis and release, which was suppressed by the COX-1 inhibitor valeryl salicylate but not by the COX-2 inhibitor NS-398. Experiments carried out by adding exogenous PGE(2) to HL-60 cells indicated that PGE(2) was not involved in TRAIL cytotoxicity and rather showed a dose-dependent protection against TRAIL-induced apoptosis. Importantly, the ability of TRAIL to increase PGE(2) production was also observed in normal, human CD34-derived myeloid cells and in freshly isolated peripheral blood CD14(+) monocytes. Moreover, in contrast to HL-60 cells, primary, normal cells were not susceptible to TRAIL cytotoxicity. These data indicate that the ability of TRAIL to up-regulate eicosanoid production and release is not ...
The presence of a secondary cartilage in the mental symphyseal region was examined in this study.... more 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.
The expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL(TNFSF10)) and of... more The expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL(TNFSF10)) and of its receptors (TRAILR1, TRAILR2, TRAILR3, and TRAILR4) have been documented in testis, but the presence of soluble TRAIL in seminal fluid, as well as the potential physiopathological role of the TRAIL/TRAILR system in spermatozoa, has not been previously investigated. Male donors (n=123) among couples presenting for infertility evaluation were consecutively enrolled in this study. The presence of soluble TRAIL was analyzed in seminal samples by ELISA, while the surface expression of TRAIL receptors was investigated by flow cytometry. High levels of soluble TRAIL were detected in seminal plasma (median, 11 621 pg/ml and mean±s.d., 13 371±8367 pg/ml) and flow cytometric analysis revealed a variable expression of TRAIL receptors in the sperm cellular fraction among different subjects. In addition, the effect of physiologically relevant concentrations of recombinant TRAIL was investigated on survival and motility of spermatozoa. Of interest, the in vitro exposure of capacitated spermatozoa to recombinant TRAIL (10 ng/ml) significantly preserved their overall survival. Therefore, the present study demonstrates for the first time the presence of elevated levels of the anti-inflammatory cytokine TRAIL in seminal fluids. Moreover, the demonstration that recombinant TRAIL promotes spermatozoa survival after capacitation suggests potential therapeutic implications.
A short-term (72-96 hours) biocompatibility evaluation in vitro of four single phase dental metal... more A short-term (72-96 hours) biocompatibility evaluation in vitro of four single phase dental metal alloys was conducted by determining cell proliferation rates correlated to the organization of the extracellular matrix protein fibronectin in human fibroblast cultures. Immunocytochemical methods were performed to detect both cell proliferation rates by 5-bromodeoxyuridine (BrdU) incorporation, and fibronectin arrangement, i.e., diffuse in the extracellular matrix, organized in fibrils or in focal adhesions. We showed that cell proliferation rates were related to fibronectin expression. In particular, a higher percentage of cells in the S-phase were related to a predominance of fibronectin organized both in fibrils and in focal adhesions. The alloy with the highest Au content seemed the most biocompatible among those tested, since it behaved in a very similar manner to the controls. On the contrary, fibroblasts exposed to the alloy with the highest percentage of Ag had the most different behavior as compared to the controls. We can assume that a correlation exists between fibronectin organization and the percentage of BrdU-positive cells and that these parameters are varying with the different metal composition of the alloys. The observation of fibronectin arrangement together with cell proliferation rates could be considered a useful tool to determine the biocompatibility of these biomaterials.
Apoptotic cell death is characterized by deep morphological changes that take place in the nucleu... more Apoptotic cell death is characterized by deep morphological changes that take place in the nucleus. It is unclear whether modifications also occur in the nuclear matrix, a mainly proteinaceous structure that conceivably acts as a nuclear framework. We have investigated whether biochemical and morphological alterations of the nuclear matrix prepared from apoptotic HL-60 cells were dependent on the manipulations to which isolated nuclei were subjected before DNase I digestion and 2 M NaCl extraction. Our results showed that the stabilizing procedures employed to preserve the inner fibrogranular network and nucleolar remnants of the matrix (i.e., a 37 degrees C incubation; exposure to sodium tetrathionate at 4 degrees C; exposure to sodium tetrathionate at 37 degrees C) had no effect on the protein recovery of apoptotic nuclear matrices, which was always approximately two- to fivefold less than in control matrices. Moreover, one- and two-dimensional gel analysis of nuclear matrix proteins showed that, in apoptotic samples, striking quantitative changes were present, as compared with controls. Once again, these changes were seen irrespective of the stabilizing procedures employed. Also, transmission electron microscope analysis showed similar morphological alterations in all types of apoptotic nuclear matrices. By contrast, the immunofluorescent distribution of the 240-kDa NuMA protein seen in apoptotic samples was more sensitive to the stabilizing treatments. Our results indicate that the biochemical and morphological changes of the apoptotic nuclear matrix are largely independent of the isolation protocols and strengthen the contention that destruction of the nuclear matrix network is one of the key events leading to apoptotic nuclear destruction.
Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell ... more 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)
Apoptosis is a form of active cell death, genetically encoded, that plays a key role during sever... more Apoptosis is a form of active cell death, genetically encoded, that plays a key role during several physiological and pathological conditions. During the apoptotic process, striking morphological and biochemical changes take place in the cell nucleus. However, the molecular mechanisms underlying these changes have escaped clarification for many years. Recently, attention has been devoted to identifying the modifications that occur during apoptosis in the nuclear matrix, a mainly proteinaceous framework structure which is thought to play a fundamental role in organizing nuclear structure and function. In this review, we focus our attention on the biochemical and morphological changes detected in the nuclear matrix during the apoptotic process. Particular emphasis will be placed on the proteolysis that some nuclear matrix proteins undergo early during the apoptotic process, as well as on the detachment of DNA loops from the matrix by the action of endonuclease(s). Future research in this field may provide important information about the principal mechanisms that cause nuclear destruction in apoptotic cells.
The effect of ICRF 159 on adriamycin (ADR) cardiotoxicity and on total myocardial calcium content... more The effect of ICRF 159 on adriamycin (ADR) cardiotoxicity and on total myocardial calcium content was examined in rats. ICRF 159 did not increase the survival time of ADR-treated animals; however the histological findings showed a significant prevention of ADR-induced cardiomyopathy (CMP) by ICRF. The total myocardial calcium content of animals treated with ADR was significantly higher, while no significant difference was seen in animals pretreated with ICRF 159 as compared with controls. As these findings suggested a role of calcium in ADR CMP and in the pharmacological action of ICRF in this disease, we also tested a closely related chelating agent, EDTA. This molecule decreased myocardial calcium levels in ADR-treated animals almost to normal values; however the histological cardiac alterations were not prevented.
Molecular targeted therapies have changed the landscape of cancer research. Agonistic monoclonal ... more Molecular targeted therapies have changed the landscape of cancer research. Agonistic monoclonal antibodies (MoAbs) targeting TRAIL-death receptors (TRAIL-Rs) have been developed and currently used in clinical trials. Binding of such antibodies to TRAIL-R1 and TRAIL-R2 results in death inducing signalling complex (DISC) formation and induction of apoptosis, which represents a natural mechanism of cell growth control and an ideal target for drug development. These novel fully humanized compounds have been associated with conventional chemotherapy in the treatment of advanced solid malignancies, including different types of lymphoma. Here we outline the rationale and potential of a new molecular-based strategy combining agonistic anti-TRAIL-death receptor monoclonal antibodies plus the pioneer of the new biological frontiers of cancer therapy: rituximab.
Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia
A triploid fetus (karyotype 69, XXX) with crown-rump length (CRL) 94 mm, presenting micro- and re... more A triploid fetus (karyotype 69, XXX) with crown-rump length (CRL) 94 mm, presenting micro- and retrognathia, low-set ears and crooked feet, was cleared and double-stained with alizarin red S and alcian blue for detecting the ossification patterns in the vertebral column, ribs, ischium, limbs, and face. Longitudinal measurements of some long bones in the upper (humerus, ulna, radius) and lower (femur, tibia, fibula) limb were taken. The values of both the total length (TL) and the ossified part (OL) of each long bone, as well as the OL/TL per cent ratio were considered. 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. Since the aim of this work was to assess the influence of triploidy 69, XXX the skeletal development and growth patterns, all values obtained in the examined specimen were related with those relative to a group of fetuses, witho...
Quintessence international (Berlin, Germany : 1985)
This short-term (72- to 96-hour) in vitro study on fibroblasts evaluated the biocompatibility of ... more This short-term (72- to 96-hour) in vitro study on fibroblasts evaluated the biocompatibility of 3 single-phase dental alloys by determining cellular proliferation rates and the expression of a glycoprotein, fibronectin, which is involved in cellular adhesion processes. Flow 2002 fibroblasts were cultured together with 3 single-phase dental alloys of different composition. Proliferation rates were determined by 5-bromodeoxyuridine incorporation. Fibronectin expression was determined by indirect immunofluorescence. At 72 hours, cells cultured with the alloy containing the lowest amount of noble elements (gold, platinum, and palladium) and the highest amount of silver exhibited significantly less proliferation than did controls. At 96 hours, only cultures with the alloy containing the greatest amount of noble elements behaved in a way similar to controls. Fibronectin organization in fibrils and in focal adhesions was correlated to higher cellular proliferation rates. Fibronectin organ...
Italian journal of anatomy and embryology = Archivio italiano di anatomia ed embriologia
In our previous work we have investigated the expression of the serine-threonine kinase protein k... more In our previous work we have investigated the expression of the serine-threonine kinase protein kinase C (PKC) in the vertebral column of mouse foetuses. In the present work we would verify the expression of four PKC-isoenzymes (alpha, delta, epsilon, zeta) in two distinct phases of the chondrogenesis and the endochondral osteogenesis in vitro. We performed primary cultures of chondrocytes collected from tibiae of 6-day old chick embryos. This cells were cultured for 20 days and than collected on coverslips (stage 1 culture). Other cells of the stage 1 were undergone further differentiation towards the phenotype of osteoblast-like cells (stage 2 culture), in accord to the protocol of Descalzi Cancedda et al. (1992). In stage 1 culture, PKC-epsilon was the most expressed isoform, whereas PKC-alpha exhibited the least intense positivity. In stage 2 culture, PKC-alpha was the most expressed isoform, whereas a marked decrease of PKC-epsilon expression was detected compared to stage 1. N...
Bollettino della Società italiana di biologia sperimentale
Recent studies have shown that protein kinase C (PKC) plays a pivotal role in many cellular funct... more Recent studies have shown that protein kinase C (PKC) plays a pivotal role in many cellular functions, i.e. cell proliferation and differentiation, exocytosis, ion-exchange regulation, hormone and neurotransmitter release, programmed cell death. Up to now the tissue and organ distribution of PKC isoenzymes have been studied in various mammalian adults and it has been suggested that each of them could play a specific role in the regulation of various cellular functions. However, few works have been performed on the expression of the enzyme in actively proliferating and differentiating tissues, i.e. during embryonal and fetal developmental stages. In the present study we have performed an immunohistochemical analysis by using polyclonal antibodies in order to verify the distribution of nine PKC isoenzymes in the vertebral column of human fetuses in a key period (8th developmental week), when the most relevant chondrogenetic and osteogenetic processes occur. The detection of the variou...
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) up-regulated the expression of co... more Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) up-regulated the expression of constitutive cyclooxygenase (COX)-1 protein in HL-60 cells without affecting COX-2. The TRAIL-mediated COX-1 up-regulation was accompanied by a significant increase of the PGE(2) synthesis and release, which was suppressed by the COX-1 inhibitor valeryl salicylate but not by the COX-2 inhibitor NS-398. Experiments carried out by adding exogenous PGE(2) to HL-60 cells indicated that PGE(2) was not involved in TRAIL cytotoxicity and rather showed a dose-dependent protection against TRAIL-induced apoptosis. Importantly, the ability of TRAIL to increase PGE(2) production was also observed in normal, human CD34-derived myeloid cells and in freshly isolated peripheral blood CD14(+) monocytes. Moreover, in contrast to HL-60 cells, primary, normal cells were not susceptible to TRAIL cytotoxicity. These data indicate that the ability of TRAIL to up-regulate eicosanoid production and release is not ...
The presence of a secondary cartilage in the mental symphyseal region was examined in this study.... more 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.
The expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL(TNFSF10)) and of... more The expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL(TNFSF10)) and of its receptors (TRAILR1, TRAILR2, TRAILR3, and TRAILR4) have been documented in testis, but the presence of soluble TRAIL in seminal fluid, as well as the potential physiopathological role of the TRAIL/TRAILR system in spermatozoa, has not been previously investigated. Male donors (n=123) among couples presenting for infertility evaluation were consecutively enrolled in this study. The presence of soluble TRAIL was analyzed in seminal samples by ELISA, while the surface expression of TRAIL receptors was investigated by flow cytometry. High levels of soluble TRAIL were detected in seminal plasma (median, 11 621 pg/ml and mean±s.d., 13 371±8367 pg/ml) and flow cytometric analysis revealed a variable expression of TRAIL receptors in the sperm cellular fraction among different subjects. In addition, the effect of physiologically relevant concentrations of recombinant TRAIL was investigated on survival and motility of spermatozoa. Of interest, the in vitro exposure of capacitated spermatozoa to recombinant TRAIL (10 ng/ml) significantly preserved their overall survival. Therefore, the present study demonstrates for the first time the presence of elevated levels of the anti-inflammatory cytokine TRAIL in seminal fluids. Moreover, the demonstration that recombinant TRAIL promotes spermatozoa survival after capacitation suggests potential therapeutic implications.
A short-term (72-96 hours) biocompatibility evaluation in vitro of four single phase dental metal... more A short-term (72-96 hours) biocompatibility evaluation in vitro of four single phase dental metal alloys was conducted by determining cell proliferation rates correlated to the organization of the extracellular matrix protein fibronectin in human fibroblast cultures. Immunocytochemical methods were performed to detect both cell proliferation rates by 5-bromodeoxyuridine (BrdU) incorporation, and fibronectin arrangement, i.e., diffuse in the extracellular matrix, organized in fibrils or in focal adhesions. We showed that cell proliferation rates were related to fibronectin expression. In particular, a higher percentage of cells in the S-phase were related to a predominance of fibronectin organized both in fibrils and in focal adhesions. The alloy with the highest Au content seemed the most biocompatible among those tested, since it behaved in a very similar manner to the controls. On the contrary, fibroblasts exposed to the alloy with the highest percentage of Ag had the most different behavior as compared to the controls. We can assume that a correlation exists between fibronectin organization and the percentage of BrdU-positive cells and that these parameters are varying with the different metal composition of the alloys. The observation of fibronectin arrangement together with cell proliferation rates could be considered a useful tool to determine the biocompatibility of these biomaterials.
Apoptotic cell death is characterized by deep morphological changes that take place in the nucleu... more Apoptotic cell death is characterized by deep morphological changes that take place in the nucleus. It is unclear whether modifications also occur in the nuclear matrix, a mainly proteinaceous structure that conceivably acts as a nuclear framework. We have investigated whether biochemical and morphological alterations of the nuclear matrix prepared from apoptotic HL-60 cells were dependent on the manipulations to which isolated nuclei were subjected before DNase I digestion and 2 M NaCl extraction. Our results showed that the stabilizing procedures employed to preserve the inner fibrogranular network and nucleolar remnants of the matrix (i.e., a 37 degrees C incubation; exposure to sodium tetrathionate at 4 degrees C; exposure to sodium tetrathionate at 37 degrees C) had no effect on the protein recovery of apoptotic nuclear matrices, which was always approximately two- to fivefold less than in control matrices. Moreover, one- and two-dimensional gel analysis of nuclear matrix proteins showed that, in apoptotic samples, striking quantitative changes were present, as compared with controls. Once again, these changes were seen irrespective of the stabilizing procedures employed. Also, transmission electron microscope analysis showed similar morphological alterations in all types of apoptotic nuclear matrices. By contrast, the immunofluorescent distribution of the 240-kDa NuMA protein seen in apoptotic samples was more sensitive to the stabilizing treatments. Our results indicate that the biochemical and morphological changes of the apoptotic nuclear matrix are largely independent of the isolation protocols and strengthen the contention that destruction of the nuclear matrix network is one of the key events leading to apoptotic nuclear destruction.
Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell ... more 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)
Apoptosis is a form of active cell death, genetically encoded, that plays a key role during sever... more Apoptosis is a form of active cell death, genetically encoded, that plays a key role during several physiological and pathological conditions. During the apoptotic process, striking morphological and biochemical changes take place in the cell nucleus. However, the molecular mechanisms underlying these changes have escaped clarification for many years. Recently, attention has been devoted to identifying the modifications that occur during apoptosis in the nuclear matrix, a mainly proteinaceous framework structure which is thought to play a fundamental role in organizing nuclear structure and function. In this review, we focus our attention on the biochemical and morphological changes detected in the nuclear matrix during the apoptotic process. Particular emphasis will be placed on the proteolysis that some nuclear matrix proteins undergo early during the apoptotic process, as well as on the detachment of DNA loops from the matrix by the action of endonuclease(s). Future research in this field may provide important information about the principal mechanisms that cause nuclear destruction in apoptotic cells.
The effect of ICRF 159 on adriamycin (ADR) cardiotoxicity and on total myocardial calcium content... more The effect of ICRF 159 on adriamycin (ADR) cardiotoxicity and on total myocardial calcium content was examined in rats. ICRF 159 did not increase the survival time of ADR-treated animals; however the histological findings showed a significant prevention of ADR-induced cardiomyopathy (CMP) by ICRF. The total myocardial calcium content of animals treated with ADR was significantly higher, while no significant difference was seen in animals pretreated with ICRF 159 as compared with controls. As these findings suggested a role of calcium in ADR CMP and in the pharmacological action of ICRF in this disease, we also tested a closely related chelating agent, EDTA. This molecule decreased myocardial calcium levels in ADR-treated animals almost to normal values; however the histological cardiac alterations were not prevented.
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