Biomedical subjects

M Mazzoni

Publications and source records attributed to M Mazzoni.

At least 19 recordsLinked to original sources

PubMed2005

Predictive value of EuroSCORE on long term outcome in cardiac surgery patients: a single institution study.

OBJECTIVES: To assess the value of the European system for cardiac operative risk evaluation (EuroSCORE), a validated model for prediction of in-hospital mortality after cardiac surgery, in predicting long term event-free survival. DESIGN AND SETTING: Single institution observational cohort study. PATIENTS: Adult patients (n = 1230) who underwent cardiac surgery between January 2000 and August 2002. RESULTS: Mean age was 65 (11) years and 32% were women. Type of surgery was isolated coronary artery bypass grafting in 62%, valve surgery in 23%, surgery on the thoracic aorta in 4%, and combined or other procedures in 11%. Mean EuroSCORE was 4.53 (3.16) (range 0-21); 366 were in the low (0-2), 442 in the medium (3-5), 288 in the high (6-8), and 134 in the very high risk group (> or = 9). Information on deaths or events leading to hospital admission after the index discharge was obtained from the Regional Health Database. Out of hospital deaths were identified through the National Death Index. In-hospital 30 day mortality was 2.8% (n = 34). During 2024 person-years of follow up, 44 of 1196 patients discharged alive (3.7%) died. By Cox multivariate analysis, EuroSCORE was the single best independent predictor of long term all cause mortality (hazard ratio (HR) 1.55, 95% confidence interval (CI) 1.03 to 2.34, p < 0.0001). In the time to first event analysis, 227 either died without previous events (n = 20, 9%) or were admitted to hospital for an event (n = 207, 91%). EuroSCORE (HR 1.60, 95% CI 1.36 to 1.89, p < 0.0001), the presence of > or = 2 co-morbidities versus one (HR 1.49, 95% CI 1.09 to 2.02, p < 0.0001), and > 96 hours' stay in the intensive care unit after surgery (HR 2.04, 95% CI 1.42 to 2.95, p = 0.0001) were independently associated with the combined end point of death or hospital admission after the index discharge. CONCLUSIONS: EuroSCORE and a prolonged intensive care stay after surgery are associated with long term event-free survival and can be used to tailor long term postoperative follow up and plan resource allocation for the cardiac surgical patient.

Aged↗

PubMed2002

Cell proliferation patterns in canine infundibular keratinizing acanthoma and well differentiated squamous cell carcinoma of the skin.

The aim of the present study was to investigate if the evaluation of cell proliferation of the well differentiated squamous cell carcinoma (WDSCC) and infundibular keratinizing acanthoma (IKA) could be useful in the differential diagnosis between these two tumours. Eighteen IKAs and ten WDSCCs were selected for this study. Two different methods were used to assess the activity of cell proliferation: MIB1 immunohistochemical detection and AgNOR proteins silver staining. The quantification of proliferative parameters was performed by means of an image analyzer and expressed as MIB1 index and AgNOR area (MNORA). Both MIBI immunohistochemical and AgNOR histochemical patterns were different in WDSCC and IKA; moreover analysis of variance showed a significant difference for both parameters employed (MIB1 index, MNORA) between WDSCC and IKA (P<0.003 for MIB1 index; P<0.0001 for AgNOR area). The results show that canine WDSCC and IKA have a different proliferative behaviour and the assessment of cell proliferation can be considered as a useful adjunctive tool to the histopathological investigation in the differential diagnosis of these tumours.

Animals↗

PubMed2001

HIV-1 Tat protein down-regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3-kinase/AKT/cyclic nucleoside phosphodiesterase pathway.

The addition of low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein to PC12 neuronal cells stimulated a rapid (peak at 5 min) elevation of the cAMP intracellular levels, which in turn induced the phosphorylation of CREB transcription factor (peak at 15 min) on serine-133 (Ser-133). On the contrary, at later time points (60-120 min) Tat induced a significant decline of intracellular cAMP with respect to the basal levels observed in control cells treated with bovine serum albumin. In blocking experiments performed with pharmacological inhibitors, Tat decreased the intracellular levels of cAMP and CREB Ser-133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3-kinase, AKT, and cyclic nucleoside phosphodiesterases. Moreover, in transient transfection experiments, Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP-responsive elements (CRE) in the CREB promoter. Consistently, the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged (24-48 h) treatment with Tat. This decline in the expression of CREB, which plays an essential role in the survival and function of neuronal cells, anticipated a progressive increase of apoptosis in Tat-treated cells. Although obtained in a neuronal cell line, our findings might help to explain some aspects of the pathogenesis of HIV-1-associated dementia.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2000

Cold rubidium molecules formed in a magneto-optical trap

We report the first observation of translationally cold ( approximately 90 &mgr;K) Rb2 molecules. They are produced in a magneto-optical trap in their triplet ground state. The detection is performed by selective mass spectroscopy after two-photon ionization into Rb+2, resonantly enhanced through the intermediate a (3)Sigma(+)(u)-->2 (3)Pi(g) molecular band. The two rubidium isotopes present very different types of behavior that are interpreted in terms of their respective collisional properties.

Journal Article↗

PubMed2000

HIV-1 Tat-mediated inhibition of the tyrosine hydroxylase gene expression in dopaminergic neuronal cells.

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein or tat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivo injection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinson's-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH(+) neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

Animals↗

PubMed1999

Regulation of the platelet serotonin transporter by protein kinase C in the young and elderly.

BACKGROUND: Some data show that different factors may influence the serotonin (5-HT) uptake rate. Our study aimed at evaluating the possible role of a protein kinase C (PKC) activator, i.e., 4-beta-12-tetradecanoylphorbol-13-acetate (beta-TPA) on the platelet 5-HT uptake of young and elderly subjects, through the measurement of the 5-HT uptake itself and 3H-paroxetine ([3H]PAR) binding sites, which correspond to the transporter protein. METHODS: Human platelets and 5-HT uptake were evaluated according to the method of Arora and Meltzer, while [3H]PAR binding was performed following the Marazziti et al method. RESULTS: The results showed that beta-TPA reduced significantly the maximal velocity (Vmax) of 5-HT uptake, with no change in the Michaelis constant or in [3H]PAR binding parameters, in platelets of both young and elderly subjects. Although this last group of subjects had a significantly lower Vmax than the other, the degree of inhibition was almost the same (75%) in both. CONCLUSIONS: These findings indicate that PKC decreases the 5-HT uptake rate by modifying the phosphorylation state of the transporter and with no change in the number of [3H]PAR binding sites. The responsiveness of this pathway is identical in both young and elderly subjects.

Adult↗

PubMed1998

HIV-1 Tat induces tyrosine phosphorylation of p125FAK and its association with phosphoinositide 3-kinase in PC12 cells.

OBJECTIVE: To evaluate the signal transduction potential of HIV-1 Tat in a neuronal cell model. METHODS: The tyrosine phosphorylation levels of the focal adhesion kinase p125FAK and its association with phosphoinositide 3-kinase (PI 3-K) were evaluated in serum-starved rat pheochromocytoma PC12 cells, either treated with low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein or stably transfected with Tat cDNA. RESULTS: Extracellular Tat induced a rapid increase of p125FAK tyrosine phosphorylation and p125FAK-associated PI 3-K activity. By using recombinant mutated Tat proteins, it was found that deletion of amino acids 73-86 encoded by the second exon of the tat gene resulted in a significant decrease of the ability of Tat to induce p125FAK tyrosine phosphorylation. Paradoxically, mutations in the basic region encoded by the first exon of tat, which is essential for nuclear localization and HIV-1 LTR transactivation, increased the ability of Tat to stimulate p125FAK tyrosine phosphorylation. Moreover, in comparison with cells transfected with a control vector, PC12 cells stably transfected with tat cDNA showed greater amounts of p125FAK protein, an increase in p125FAK tyrosine phosphorylation and higher levels of p125FAK-associated PI 3-K activity. The addition of anti-Tat neutralizing antibody to tat-transfected PC12 cells in culture blocked both the p125FAK tyrosine phosphorylation and its association with PI 3-K but did not affect the total amount of p125FAK. CONCLUSION: HIV-1 Tat protein enhanced both the expression and the functionality of p1 25FAK in PC12 neuronal cells. Whereas the first event required intracellular Tat, the increased p125FAK phosphorylation was strictly dependent upon extracellular Tat.

Animals↗

PubMed1996

Extracellular human immunodeficiency virus type-1 Tat protein activates phosphatidylinositol 3-kinase in PC12 neuronal cells.

We have here investigated the effect of the regulatory Tat protein of the human immunodeficiency virus type 1 (HIV-1) on the PI 3-kinase catalytic activity in PC12 rat pheochromocytoma cells. After as early as 1 min from the beginning of the treatment with recombinant HIV-1 Tat protein, a significant increase in the tyrosine phosphorylation levels of the p85 regulatory subunit of PI 3-kinase was noticed in 48 h serum-starved PC12 cells. Moreover, the addition of Tat to PC12 cells induced a great increase in PI 3-kinase immunoprecipitated with an anti-phosphotyrosine antibody with a peak of activity (19-fold increase with respect to the basal levels) after a 15-min treatment. This increase in PI 3-kinase activity was significantly higher in PC12 cell cultures supplemented with Tat protein than in cultures stimulated by 100 ng/ml nerve growth factor (NGF; 8-fold increase with respect to the basal levels). Further experiments showed that Tat protein was able to specifically activate PI 3-kinase at picomolar concentrations. In fact: (i) maximal activation of PI 3-kinase was observed at concentrations as low as 1 ng/ml and was specifically blocked by anti-Tat neutralizing antibody; (ii) a Tat-dependent activation was also observed in experiments in which PI 3-kinase activity was evaluated in either anti-Tyr(P) or anti-p85 immunoprecipitates; (iii) 100 nM wortmannin completely blocked the Tat-mediated increase in PI 3-kinase activity both in vitro and in vivo. Our data strongly support the concept that extracellular Tat acts as a cell stimulator, inducing intracellular signal transduction in uninfected cells.

Animals↗

PubMed1996

Changes of nuclear protein kinase C activity and isotype composition in PC12 cell proliferation and differentiation.

To establish whether protein kinase C was involved in the nuclear events underlying cell differentiation and proliferation, rat pheochromocytoma PC12 cells, serum-starved for 24 h, were treated with either differentiating doses of nerve growth factor or high serum concentrations, which represented a powerful mitogenic stimulus. Western blot analysis with isoform-specific antibodies, performed on whole cell homogenates, cytoplasms, and purified nuclei, showed that PKC isotypes alpha, beta I, beta II, delta, epsilon, eta, and zeta were expressed in PC12 cells and that all of them, except for beta I, were found at the nuclear level, variably modulated depending on the cell treatment. Compared to serum-stimulated cells, in which an early (1 day) and marked rise of protein kinase C activity was followed by a plateau, nerve growth factor-treated cells showed a progressive increase of protein kinase C activity coincident with the onset and maintenance of the differentiated phenotype. Western blot analysis of nuclei isolated from fully differentiated cells demonstrated an increase of protein kinase C alpha, paralleled by enhanced phosphotransferase activity along with the nerve growth factor treatment, and complete loss of the delta isotype. In contrast, in nuclei of proliferating PC12 cells, after an early but modest increase at 1 day of mitogenic stimulation, protein kinase C activity reached a plateau. Isotype-specific analysis indicated a concomitant increase of protein kinase C beta II, delta, and zeta and the appearance of protein kinase C epsilon and eta at the nuclear level. Considering the relative intensity of the cytoplasmic and nuclear immunoreactive bands under the three conditions examined, clear-cut translocation to the nucleus occurred for PKC epsilon and eta in serum-stimulated cells. Additional nuclear accumulation of PKC by translocation from the cytoplasm was prominently induced for the zeta isoform after mitogenic stimulation and for PKC alpha during prolonged NGF treatment. Our data suggest that nuclear translocation and selective activation of distinct protein kinase C isoforms play a relevant role in the control of proliferation and differentiation of the same cell type and that nuclear protein kinase C is crucial to the induction and persistence of the differentiated neuronal phenotype of PC12 cells.

Amino Acid Sequence↗

PubMed1996

Primary progressive aphasia: description of a clinical case with nine years of follow-up.

We describe the case of a woman with primary progressive aphasia who, over a period of nine years, has shown no signs of clinical deterioration and has only a symptomatic language disturbance. Neuropsychological follow-up has revealed progressive language impairment, with the integrity of praxis, visuoperceptive gnosia and short term visuospatial memory remaining intact; the only impairment was that revealed by long-term memory tests.

Aged↗

PubMed1996

Intraoperative phrenic nerve monitoring in cardiac surgery.

BACKGROUND: Left hemidiaphragmatic paralysis due to phrenic nerve lesion is a frequent complication of hypothermic cardiopulmonary bypass. Although this is believed to be caused by cold injury to the phrenic nerve, its exact cause is still not clear. STUDY OBJECTIVE: To assess feasibility, safety, and usefulness of intraoperative phrenic nerve function monitoring. SETTING: Elective cardiac surgery in a university hospital. PATIENTS: Consenting patients scheduled for myocardial revascularization surgery with the use of the left internal mammary artery. DESIGN: Intraoperative monitoring of compound diaphragmatic action potentials (CDAPs) through transcutaneous stimulation of phrenic nerves. INTERVENTIONS: Patients were divided in two groups. Group 1 received intracoronary cold St. Thomas's solution as the only cardioplegic method. Group 2 received topical cardiac cooling with ice-cold solutions in addition to intracoronary cardioplegia. RESULTS: In all group 1 patients, function of phrenic nerves was maintained throughout the surgical procedure. Group 2: in two patients, bilateral, and in one patient, left phrenic nerve conduction was abolished after submersion of the heart in ice-cold solution. In two of them, the action potential of the left hemidiaphragm was absent by the end of surgery. In one, nerve conduction recovered with rewarming of the patient. DISCUSSION: Intraoperative monitoring of CDAP was safe and easily obtained in the intraoperative setting. It allowed us to observe changes in phrenic nerve conduction occurring during surgery and as a result of cold cardioplegia. Cryogenic lesion of phrenic nerve might explain our findings. However, nerve ischemia cannot be ruled out and it may worsen axonal damage or delay its recovery. COMMENT: This monitoring method allowed us to predict postoperative diaphragmatic dysfunction. Also, surgeons can be warned of the damaging effects of excessive cooling of the pericardium and surrounding structures; thus, preventive measures can be taken.

Action Potentials↗

PubMed1995

The human immunodeficiency virus type-1 Tat protein upregulates Bcl-2 gene expression in Jurkat T-cell lines and primary peripheral blood mononuclear cells.

The regulatory Tat protein of human immunodeficiency virus type-1 (HIV-1) exerts a pleyotropic activity on the survival and proliferation of different cell types in culture. In this report, we investigated the effect of either endogenous or exogenous Tat on Bcl-2 proto-oncogene expression and cell survival in Jurkat T-cell lines and primary peripheral blood mononuclear cells. Stable and transient transfections of Jurkat cells with the cDNA of tat and a plasmid containing Bcl-2 promoter in front of CAT (Bcl-2 Pr/CAT) stimulated CAT activity and showed an increase of Bcl-2 mRNA and protein expression. This effect was specifically related to tat, because Jurkat cells transfected with the cDNA of tat in antisense orientation, tat carrying a mutation in the amino acid cys22-gly22, or the control vector alone (pRPneo-SL3) did not show any significant difference in Bcl-2 promoter activity with respect to parental Jurkat cells. We also observed a specific correlation between tat-induced Bcl-2 gene expression and inhibition of apoptosis induced by serum withdrawal. Our results suggest that the structural integrity of the activation domain of Tat was required for the promotion of the Bcl-2 promoter and Jurkat cell survival, because a single mutation in the aminoacid cys22 was sufficient to completely block the upregulation of Bcl-2 and inhibition of apoptosis. Moreover, picomolar concentrations of native or recombinant Tat were able to upregulate Bcl-2 expression both in Jurkat and primary peripheral blood mononuclear cells, suggesting that extracellular Tat, actively released by infected cells, may also play a significant role in suppressing apoptosis. An aberrant cell survival of lymphoid cells consequent to the upregulation of Bcl-2 may represent an additional pathogenetic mechanism that could help explain both the dysregulated immune response and the frequent occurrence of hyperplastic/neoplastic disorders in HIV-1-seropositive individuals.

Apoptosis↗

PubMed1995

An autocrine loop of HIV type-1 Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal HIV-1 LTR transactivating activity.

Human immunodeficiency virus type 1 (HIV-1) transactivating Tat protein is pivotal to virus replication. Tat's potential effects on HIV-1 pathogenesis, however, go well beyond its role in the virus's life cycle. Current data indicate that biologically active Tat is released from HIV-1-infected cells and readily endocytosed and targeted to the nucleus of nearby, or perhaps distant, cells, where it may exert a series of pleiotropic effects. This paracrine action has been extensively investigated, and depending on the amounts of exogenously added Tat, its effects may extend from the suppression of immunocompetent cells to transactivation of heterologous genes to the promotion of growth of Kaposi's sarcoma spindle cells. We have already observed that various cell lines, either permanently transfected with an expressive HIV-1 tat gene construct or cultured in the presence of exogenously added Tat protein, are protected from programmed cell death after serum withdrawal or other apoptotic stimuli. The present article shows that various types (lymphoblastoid, epithelial, neuronal) of permanently tat-transfected cell lines actively release fully bioactive Tat protein. The addition of anti-Tat antibody to the culture medium completely abolishes their increased survival/proliferation capacity in serum-free culture. In these conditions, therefore, the enhanced survival/proliferation potential of permanently tat-transfected cells seems entirely dependent on a Tat-protein autocrine loop. The finding that anti-Tat antibody, added to culture medium, exerts a negative influence on the expression of a Tat-responsive HIV-1 long terminal repeat chloramphenicol-acetyltransferase construct, transiently transfected into permanently tat-transfected cells, suggests that the Tat autocrine loop may also be required for optimal HIV-1 long terminal repeat transactivation.

Animals↗

PubMed1995

Identification of PI-PLC beta 1, gamma 1, and delta 1 in rat liver: subcellular distribution and relationship to inositol lipid nuclear signalling.

The subcellular distribution of PI-PLC beta 1, gamma 1, and delta 1 has been investigated in rat liver by western blot and immunohistochemical analysis with a panel of isoform-specific antibodies. The data obtained in situ on cryo-sectioned tissue indicate that PI-PLC beta 1 is predominantly nuclear, while gamma 1 is largely cytoplasmic and delta 1 is sharply restricted to the cytoplasm. In fractionation experiments, the Western blot analysis indicated that the recovery of the nuclear isoforms beta 1 and gamma 1 was not affected by the removal of the nuclear membrane, and that the two enzymes persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent. The assay of the phosphodiesterase activity in different cell fractions correlates with the observed relative abundance of the enzymes, and specific inhibition with neutralizing anti-beta 1 and -gamma 1 isoforms confirms that these are the enzymes active at the nuclear level. These results demonstrate that in rat liver cells, as in other cell types, different members of the PI-PLC family show a discrete intracellular distribution, and suggest that PI-PLC beta 1 and gamma 1 play a central role in modulating the nuclear phosphoinositide cycle.

Animals↗

PubMed1995

Increased sinusoidal wall permeability and liver fatty change after two-thirds hepatectomy: an ultrastructural study in the rat.

Accumulation of lipids in the hepatocyte cytoplasm after partial hepatectomy (PH) has long been recognized, but the mechanism behind this phenomenon is still poorly understood. In this study, rats subjected to a standard two-thirds PH showed early and marked increase in portal venous pressure (P < .01). On scanning electron microscopy, the regenerating liver fixed by portal perfusion under hemodynamic conditions identical to that found in vivo during the first 24 hours showed a significant (P < .01) 10-fold increase in the sinusoidal wall porosity (percentage open area by fenestrations). This was paralleled by the disappearance of the sieve-plate arrangement of small fenestrations and by a significant decrease in the number of fenestrations per micrometers squared of sinusoidal surface at 6 (P < .01) and 12 hours (P < .05). In addition, there were major changes in the frequency and distribution of all three classes of fenestrations. At 6 and 12 hours, there was a marked decrease of small class 1 fenestrations and a marked increase of intermediate class 2 fenestrations and large class 3 fenestrations (P < .0001). A concurrent accumulation of lipid droplets in the hepatocyte cytoplasm produced a 20-fold increase in the hepatocyte total lipid volume. A statistically significant linear correlation (r = 0.907; P < .01) was found between the amount of intracellular lipids and the data quantitating the changes in porosity of liver sinusoids at 24 hours. It is concluded that an increased sinusoidal wall permeability to lipids may be the primum moves in the pathogenesis of transient liver fatty change after PH in the rat.

Animals↗

PubMed1994

Lactulose hydrogen breath test in orocecal transit assessment. Critical evaluation by means of scintigraphic method.

Orocecal transit time can be studied easily using the hydrogen breath test with lactulose, but the method has some important limitations. The orocecal transit time of 10 patients suffering from irritable bowel syndrome was measured twice, at a one-week interval, by breath test and scintigraphy simultaneously using an aqueous solution of 20 g lactulose containing 74 MBq of [99mTc]DTPA. Abdominal radioactivity and alveolar hydrogen values obtained every 5 min were noted and used to obtain the following: orocecal transit time by the two methods; ileocecal lactulose flow; total and per gram of lactulose hydrogen production; mean hydrogen concentration during the right colon filling; and measurement error of the breath test with respect to the scintigraphy. In the case of the breath test, the orocecal transit time intrapatient reproducibility was better (coefficient of variation = 13.5%) when a hydrogen threshold increment of 5 ppm was used; the best correlation with the scintigraphic measurement was observed at this threshold (r = 0.90, P < 0.001). The breath test overestimated orocecal transit time with the error correlating negatively and significantly with the total hydrogen production and, particularly, the mean hydrogen concentration (r = 0.79, P < 0.01): for a mean hydrogen concentration of more than 15 ppm, the error was negligible, while within this value there was a noticeable overestimation. To conclude, the lactulose hydrogen breath test is capable of giving an accurate measurement of orocecal transit time if a hydrogen threshold increment of 5 ppm is chosen and if the mean hydrogen concentration in the first 30 min of the right colon filling is taken into account.

Adult↗

PubMed1994

Diacylglycerol kinase activity in rat liver nuclei.

Membrane-depleted rat liver nuclei contain diacylglycerol (DAG) kinase showing a specific activity which doubles that of the whole homogenate. In contrast, cytoplasmic and plasma membrane marker enzymes attain a specific activity of 0.4% at the most, when nuclear DAG kinase approaches 4.5% of the total tissue activity. The enzyme shows a Km of 161 and 200 microM for ATP in both nuclei and microsomes whereas the Km for DAG is 75 microM in nuclei and 658 microM in microsomes. Octylglucoside, CHAPS and Triton X-100 behave mainly as inhibitors, while deoxycholate stimulates the enzyme activity in both cellular fractions, increasing specific activity (3.2-fold in nuclei and 29.1-fold in microsomes) and decreasing Km for DAG (39 microM in nuclei and 237 microM in microsomes). Phospholipids and ceramide stimulate the enzyme activity in isolated nuclei, while no effect occurs in the microsomal fraction. At variance, sphingosine behaves as an inhibitor in both cellular fractions. DAG kinase also utilizes endogenous substrates mobilized by Bacillus cereus phospholipase C, which hydrolyses nuclear phosphatidylcholine and phosphatidylethanolamine and by phosphatidylinositol-specific phospholipase C, which hydrolyses nuclear PI and PIP. These data indicate that nuclear DAG can be controlled by converting it into phosphatidic acid by the action of a nuclear enzyme and support the contention that protein kinase C activity can be modulated at the nuclear level by a discrete system involving phospholipase C and DAG kinase that could operate independently from the cytoplasm.

Animals↗

PubMed1994

Immunocytochemical detection of the specific association of different PIC isoforms with cytoskeletal and nuclear matrix compartments in PC12 cells.

The increasing evidence of discrete roles of phosphoinositidase C (PIC) isoforms and the assessment of their localization in the cytoskeleton and in the nucleus support the involvement of particular isotypes of this enzyme in signal transduction at multiple levels. PC12 rat pheochromocytoma is one of the few cell lines expressing three immunologically distinct isoforms of PIC. We have analyzed the subcellular distribution of the PIC beta 1, gamma 1 and delta 1 isoforms using confocal and electron microscope immunocytochemistry. PIC beta 1 is mainly found in the nucleus and is associated with interchromatin domains. On the other hand, the PIC gamma 1 isoform is found in the nucleus and in the cytosol, while PIC delta 1 is exclusively cytoplasmic. Immunoblot and immunocytochemical experiments indicate that the various PIC isoforms are differently bound to structural cell compartments, such as cytoskeletal and nuclear matrix elements. In fact, PIC beta 1 and PIC gamma 1 isoforms are tightly associated with the nuclear matrix, while only about 50% of PIC gamma 1 is associated with the cytoskeleton after DNase I and high salt extractions. PIC gamma 1 is almost completely soluble under these conditions. These results further confirm the complexity of the inositide signal transduction mechanism, which involves several PIC isoforms, specifically localized in different cell compartments and support the existence of a membrane-unrelated inositol lipid-dependent signalling in the nuclear interior.

Animals↗