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C Giardina

Publications and source records attributed to C Giardina.

At least 19 recordsLinked to original sources
PubMed2001

NF-kappaB is involved in the survival of cerebellar granule neurons: association of IkappaBbeta [correction of Ikappabeta] phosphorylation with cell survival.

The NF-kappaB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c-Rel. NF-kappaB activity is tightly controlled by IkappaB proteins which bind to NF-kappaB preventing its translocation to the nucleus. Activation of NF-kappaB is most often mediated by IkappaB degradation, which permits NF-kappaB to enter the nucleus. We investigated the role of NF-kappaB in the survival of cerebellar granule neurons. We found that survival of these neurons in high potassium medium is blocked by three separate inhibitors of NF-kappaB activity: SN-50, N-tosyl-L-phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF-kappaB is required for neuronal survival. Gel-shift assays reveal three complexes that bind to the NF-kappaB binding site in high potassium medium. Switching these cultures to low potassium medium, a stimulus that leads to apoptotic death, causes a reduction in the level of the largest complex, which contains p65. Overexpression of p65 by transfection inhibits low potassium-induced apoptosis, whereas overexpression of IkappaBalpha promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IkappaBalpha and IkappaBbeta is altered by low potassium treatment. Similarly, no changes are seen in the nuclear or cytoplasmic levels of p50, p52, RelB and c-Rel. Phosphorylation of p65, which can lead to its activation, is unchanged. Phosphorylation of IkappaBbeta is, however, reduced by low potassium treatment. Besides being necessary for high potassium-mediated neuronal survival, NF-kappaB is also involved in the survival-promoting effects of IGF-1 and cAMP as judged by the ability of SN-50 to inhibit the actions of these survival factors and the ability of these factors to inhibit the low potassium-induced alterations in the DNA-binding activity of NF-kappaB. Taken together, our results show that NF-kappaB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF-kappaB-mediated survival of cerebellar granule neurons.

Animals↗
PubMed2000

NF-kappa B regulates transcription of the mouse telomerase catalytic subunit.

Expression of the telomerase catalytic subunit (TERT) is the rate-limiting determinant of telomerase activity in most cells. Analysis of the mouse TERT promoter revealed a potential NF-kappaB binding site 350 base pairs upstream from the translational start site. An oligonucleotide from this region of the TERT promoter bound to proteins in a nuclear extract prepared from a mouse hepatoma cell line. These proteins were identified as NF-kappaB by a number of criteria: 1) the protein complex formed on the TERT oligonucleotide had an electrophoretic mobility similar to that formed on an NF-kappaB consensus oligonucleotide; 2) protein binding to this site was enhanced by NF-kappaB activators tumor necrosis factor-alpha, phorbol 12-myristate 13-acetate, and interleukin-1beta; and 3) the complex was specific and could be supershifted with antibodies against the p50 or p65 NF-kappaB subunits. The NF-kappaB binding site from the mouse TERT promoter activated transcription when fused to a basal SV40 promoter and enhanced the activity of the native TERT promoter in mouse hepatoma cells stimulated with phorbol 12-myristate 13-acetate. Transcriptional activation by the TERT NF-kappaB site could also be enhanced by co-transfection with an NF-kappaB1 expression vector. NF-kappaB may therefore contribute to the activation of TERT expression observed in mouse tissue.

Animals↗
PubMed2000

Augmented humoral immune function in metallothionein-null mice.

Stress response proteins can play integral roles as modulators of cellular function and can be involved in mechanisms that are important to immune function. Metallothionein (MT), a cysteine-rich stress response protein, has been shown to play numerous roles in the cell: it serves as a reservoir of essential heavy metals, it scavenges free radicals, and it can sequester heavy metals. These various functions suggest that MT may also participate in modulating immune responses. In previous work, we have shown that exogenous metallothionein can suppress the developing humoral immune response when coinjected with antigen. The present study was designed to evaluate the effects of endogenous MT on the development of humoral immunity. We compared the humoral immune function of animals with a targeted disruption of Mt-1 and -2 genes (MTKO) and their wild-type counterparts. MTKO mice displayed a significantly higher humoral response to challenge with ovalbumin (OVA) compared to wild-type controls. The secondary anti-OVA response in MTKO mice is as much as 58% higher than the response in control mice injected at the same time. Overall circulatory immunoglobulin levels are also substantially higher in MTKO mice (0.039 mg/ml IgM and 0.42 mg/ml IgG) than wild-type controls. MTKO mice displayed increased B cell differentiation following OVA challenge and an enhanced lymphoproliferative response to mitogenic stimulation. These changes in immune functional capacity occur in the context of changes in the makeup of the lymphoid compartments of the blood and spleen. There are substantially fewer T and B cells in the circulation of MTKO mice, but more T cells in the spleen of these mice than in control animals. Finally, we have found that splenocytes from MTKO animals displayed significantly elevated levels of NF-kappaB activity compared to wild-type controls. In conclusion, we have provided evidence that endogenous metallothionein can modulate the immune response in vivo and that intracellular MT may modulate immune function by regulation of transcription factor activity.

Animals↗
PubMed2000

Finite thermal conductivity in 1D lattices

We discuss the thermal conductivity of a chain of coupled rotators, showing that it is the first example of a 1D nonlinear lattice exhibiting normal transport properties in the absence of an on-site potential. Numerical estimates obtained by simulating a chain in contact with two thermal baths at different temperatures are found to be consistent with those based on linear response theory. The dynamics of the Fourier modes provides direct evidence of energy diffusion. The finiteness of the conductivity is traced back to the occurrence of phase jumps. Our conclusions are confirmed by the analysis of two variants of this model.

Journal Article↗
PubMed2000

IkappaBbeta-related proteins in normal and transformed colonic epithelial cells.

The transcription factor nuclear factor-kappaB (NF-kappaB) regulates genes that can influence cell proliferation, apoptosis, and inflammatory responses. Since these events can contribute to carcinogenesis, we examined the expression of NF-kappaB inhibitory proteins (IkappaBs) in normal and transformed colonic epithelial cells. Immunohistochemical analysis of the mouse colon revealed a high level of IkappaBbeta expression in epithelial cells relative to the rest of the tissue, whereas IkappaBalpha was found primarily in cells of the lamina propria. Mouse colon tumors showed a similar cell-specific staining pattern. Immunoblot analysis of IkappaBbeta from mouse colonocytes and the human HT-29 colon cancer cell line indicated that most of the IkappaBbeta in these cells was similar to the C-terminal-truncated IkappaBbeta2 isoform. Cell fractionation studies were consistent with IkappaBbeta being a major regulator of p65-p50 NF-kappaB complexes in HT-29 cells. Interestingly, two larger proteins specifically recognized by IkappaBbeta antibodies (p106 and p112) were found in HT-29 cells and in colon tissue of carcinogen-exposed mice. The p106 and p112 proteins bound to NF-kappaB, and their levels changed during the transient interleukin-1beta activation of NF-kappaB in HT-29 cells. Evidence was obtained indicating that p106 and p112 are stably ubiquitinated forms of IkappaBbeta. We propose that deficiencies in the proteasomal degradation of IkappaBbeta lead to p106 and p112 accumulation, which in turn alter NF-kappaB regulation in colon cancer cells.

Animals↗
PubMed2000

The luminal short-chain fatty acid butyrate modulates NF-kappaB activity in a human colonic epithelial cell line.

BACKGROUND & AIMS: The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in regulating immune and inflammatory responses. Because butyrate deficiency has been associated with inflammatory bowel disease, we examined the effect of butyrate on NF-kappaB activity in the human HT-29 colonic cell line. METHODS: The influence of butyrate (4 mmol/L) on NF-kappaB activity was determined using the gel mobility shift assay. The effect of butyrate on the expression of NF-kappaB subunits and inhibitory proteins was determined by immunoblotting. NF-kappaB-regulated gene expression was assayed by primer extension of intercellular adhesion molecule 1 and Mn superoxide dismutase messenger RNA, and by analysis of a transfected luciferase reporter. RESULTS: Exposure of HT-29 cells to butyrate eliminated their constitutive NF-kappaB, p50 dimer activity. This inhibition corresponded with a reduction in p50 nuclear localization, without a reduction in expression. Butyrate also selectively modulated activation of NF-kappaB, suppressing its activation by tumor necrosis factor alpha and phorbol ester more than 10-fold, without affecting the activity induced by interleukin (IL)-1beta. Butyrate did, however, enhance formation of the stronger p65-p50 transcriptional activator in IL-1beta-stimulated cells. The changes in NF-kappaB activation did not correlate with changes in IkappaBalpha levels. Gene expression reflected DNA binding. The influence of butyrate on NF-kappaB may result in part from its ability to inhibit deacetylases because the specific deacetylase inhibitor trichostatin A has a similar effect. CONCLUSIONS: These findings suggest that the influences of butyrate on colonic inflammatory responses may result in part from its influence on NF-kappaB activation. This activity of butyrate apparently involves its ability to inhibit deacetylases.

Acetylation↗
PubMed2000

Regulation of ICAM-1 expression in mouse macrophages.

In a mouse model of silica (SI) induced lung injury, SI exposure increases expression of intercellular adhesion molecule-1 (ICAM-1) on lung (alveolar/interstitial) macrophages and alveolar type II epithelial cells. To investigate the regulation of SI induced ICAM-1 expression on mouse macrophages, freshly isolated macrophages (alveolar, peritoneal) and macrophage cell lines (MH-S, RAW 264.7) were evaluated for ICAM-1 expression elicited by the particle silica (alpha quartz; 20 microg/ml; 6 microg/cm2) or the inflammatory cytokine, TNFalpha (20 ng/ml). TNFalpha significantly increased ICAM-1 expression in all cell types whereas SI elicited an increase in peritoneal macrophages (PM) and the cell line, MH-S. This pattern of increased expression was confirmed by immunocytochemistry. To investigate the regulation of ICAM-1 expression, PM were incubated with SI, TNFalpha or media concomitantly with anti-TNFalpha antibody, the antioxidant, NAC, or the iNOS synthase inhibitor, L-NAME. Both anti-TNFalpha and NAC, but not L-NAME, inhibited elicited (TNFalpha, SI) as well as constitutive (media) ICAM-1 expression. These data demonstrate that both inflammatory cytokines and inorganic particles can increase ICAM-1 expression on mouse macrophages and that this expression is mediated, in part, by TNFalpha and reactive oxygen species.

Acetylcysteine↗
PubMed2000

Acetaminophen inhibits NF-kappaB activation by interfering with the oxidant signal in murine Hepa 1-6 cells.

A toxic dose of acetaminophen (APAP) reduces the activity of NF-kappaB in mouse liver. NF-kappaB inactivation may be important for APAP toxicity, as this transcription factor can play a central role in maintaining hepatic viability. We recently reported that APAP likewise inhibits serum growth factor activation of NF-kappaB in a mouse hepatoma cell line (Hepa 1-6 cells). Here we present evidence that APAP's antioxidant activity may be involved in this NF-kappaB inhibition in Hepa 1-6 cells. Like the antioxidants N-acetylcysteine (NAC) and pyrrolidinedithiocarbamate (PDTC), APAP was found to suppress the H(2)O(2)-induced oxidation of an intracellular reactive oxygen species probe (dihydrodichlorofluorescein) in Hepa 1-6 cells. Treatment of Hepa 1-6 cells with H(2)O(2) was sufficient for NF-kappaB activation and IkappaBalpha degradation, and APAP was able to block both of these events. The APAP inhibition of NF-kappaB activation by serum growth factors may also be due to APAP's antioxidant activity, as the antioxidants NAC and PDTC likewise inhibit this activation. The potential role of NF-kappaB and oxidant-based growth factor signal transduction in APAP toxicity is discussed.

Acetaminophen↗
PubMed2000

Transcription factor NF-kappaB participates in regulation of epithelial cell turnover in the colon.

The transcription factor nuclear factor (NF)-kappaB regulates the expression of genes that can influence cell proliferation and death. Here we analyze the contribution of NF-kappaB to the regulation of epithelial cell turnover in the colon. Immunohistochemical, immunoblot, and DNA binding analyses indicate that NF-kappaB complexes change as colonocytes mature: p65-p50 complexes predominate in proliferating epithelial cells of the colon, whereas the p50-p50 dimer is prevalent in mature epithelial cells. NF-kappaB1 (p50) knockout mice were used to study the role of NF-kappaB in regulating epithelial cell turnover. Knockout animals lacked detectable NF-kappaB DNA binding activity in isolated epithelial cells and had significantly longer crypts with a more extensive proliferative zone than their wild-type counterparts (as determined by proliferating cell nuclear antigen staining and in vivo bromodeoxyuridine labeling). Gene expression profiling reveals that the NF-kappaB1 knockout mice express the potentially growth-enhancing tumor necrosis factor (TNF)-alpha and nerve growth factor-alpha genes at elevated levels, with in situ hybridization localizing some of the TNF-alpha expression to epithelial cells. TNF-alpha is NF-kappaB regulated, and its upregulation in NF-kappaB1 knockouts may result from an alleviation of p50-p50 repression. NF-kappaB complexes may therefore influence cell proliferation in the colon through their ability to selectively activate and/or repress gene expression.

Alkaline Phosphatase↗
PubMed2000

Real-time quantification of the proliferative state in astrocytomas.

OBJECTIVE: To evaluate proliferative activity in a set of gliomas and to compare the quantitative data obtained by a real-time processor with the labelling index (LI) and mitotic index (MI). STUDY DESIGN: Ki-67 immunostaining was performed on paraffin-embedded specimens from 42 cases of glioblastomas, 17 cases of anaplastic astrocytomas and 14 cases of low grade astrocytomas. Nuclear positivity was calculated as LI and by a real-time image processor for quantitative evaluation. MI was also calculated at 10 high-power fields. The data obtained from glioblastomas were compared with those from anaplastic and low grade astrocytomas. To all the data was applied the Pearson test to verify the correlation between counting and quantitative values and between proliferative markers and survival. RESULTS: A positive trend from low grade astrocytomas to glioblastomas was found for Ki-67 (LI and quantitative values) and MI, with highly significant differences between the three grades of gliomas considered. A good correlation between LI and quantitative values of Ki-67 was found. Very little relationship resulted between survival and Ki-67 LI. No relationship was found between survival and quantitative values of Ki-67. CONCLUSION: Ki-67 allowed effective separation of astrocytic tumors with different grades of malignancy. Quantitative evaluation of color information by means of a real-time processor proved to be a useful, objective and fast way to obtain readings, useful for grading purposes but not for prognostic evaluation.

Astrocytoma↗
PubMed1999

NSAIDs and butyrate sensitize a human colorectal cancer cell line to TNF-alpha and Fas ligation: the role of reactive oxygen species.

The nonsteroidal antiinflammatory drugs (NSAIDs) indomethacin and salicylic acid and the short chain fatty acid butyrate are effective colon cancer chemopreventive agents that increase reactive oxygen species (ROS) generation in colon cancer cells. Here we demonstrate that these agents sensitize the normally resistant human HT-29 colon cancer cell line to apoptosis induced by TNF-alpha or a Fas ligating antibody. The role of ROS in this sensitization is supported by the finding that direct exposure of the cells to H2O2 is sufficient for sensitization. Neither TNF-alpha nor Fas ligation alter basal or chemopreventive agent-activated ROS generation, suggesting that the death ligands and chemopreventive agents act in a complementary fashion. The dual chemopreventive agent/death ligand treatments do not increase Fas, TNF receptor 1, Bak or c-myc expression (although salicylic acid moderately induces of Fas expression). Cell death does correlate with alterations in NF-kappa B activity: the NSAIDs, butyrate and H2O2 enhance c-Rel complex formation by TNF-alpha and provide an overall enhancement of NF-kappa B activation by Fas. The antioxidant N-acetylcysteine (NAC) blocks cell death and NF-kappa B activation induced by Fas ligation, suggesting a potential role for NF-kappa B in Fas-induced apoptosis in these cells. The effects of NAC on TNF-alpha-induced cell death are more complex, with NAC being marginally protective and itself enhancing the formation of c-Rel containing complexes at higher concentrations (25 mM). The influence of NSAIDs and butyrate on ROS generation and death ligand sensitivity may be relevant to their ability to suppress colon carcinogenesis.

Acetylcysteine↗
PubMed1999

Modulation of serum growth factor signal transduction in Hepa 1-6 cells by acetaminophen: an inhibition of c-myc expression, NF-kappaB activation, and Raf-1 kinase activity.

Acetaminophen (APAP) is a widely used analgesic and antipyretic that can lead to severe liver damage when taken at excessive doses. APAP toxicity results when cytochrome P450-generated APAP metabolites trigger an oxidative stress and covalently modify target proteins. APAP has also been reported to inhibit cells from completing S-phase through a cytochrome P450-independent mechanism, raising the possibility that APAP may directly suppress liver regeneration and repair. Here we show that APAP also inhibits entrance of Hepa 1-6 cells into the cell cycle by blocking a number of events associated with the G0-G1 transition. We have found that APAP inhibits serum growth factor activation of c-myc expression, NF-kappaB DNA binding, and Raf kinase. Therefore, the ability of APAP to inhibit passage of cells through both G1 and S phases might interfere with organ regeneration and thus exacerbate acute liver damage caused by APAP.

Acetaminophen↗
PubMed1998

Nonsteroidal anti-inflammatory drugs, short-chain fatty acids, and reactive oxygen metabolism in human colorectal cancer cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and short-chain fatty acids are effective suppressors of colorectal cancer that may work in part by accentuating apoptosis of transformed cells. Since reactive oxygen species (ROS) can play an important role in regulating cell growth and cell death, we determined the effect of the NSAIDs indomethacin and salicylic acid, and the short-chain fatty acids butyrate and propionate on ROS metabolism in the HT-29 human colorectal carcinoma cell line. We find that all of these agents increase cellular peroxide generation, as determined by two independent assays. Arachidonic acid was also found to increase ROS generation, and could synergize with indomethacin in this reaction. The NSAIDs and short-chain fatty acids under study all possess a carboxyl group, and this carboxyl group is essential for salicylic acid's ability to increase ROS production. Although the two NSAIDs examined increase peroxide production, they were both found to suppress superoxide generation by vitamin K3 (menadione), a redox cycling compound similar to those found in the colon. The short-chain fatty acids did not have this activity. The ability of these NSAIDs and short-chain fatty acids to alter cellular ROS metabolism may contribute to their chemopreventive activity.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

[Malignant mesothelioma of the pleura. Morphometric study].

Malignant mesothelioma is difficult to distinguish from other pleural malignancies and also from benign mesothelial lesions. A morphometric study has been performed to distinguish between them using quantitative size and shape parameters. Seven cases of malignant mesothelioma, 5 cases of pleural metastatic adenocarcinoma and 4 cases of benign mesothelial lesions were selected and subjected to S.A.M. (Shape Analytical Morphometry). The results, statistically evaluated, showed that morphometric parameters can be proposed for diagnostic purposes being useful in the discrimination among the three populations. In fact, multivariate discriminant analysis (MDA) of the quantitative parameters obtained by morphometrical study distinguished the three groups of lesions with only 2% of error between BML/MM, 7% of error between BML/MA and 25% between MM/MA.

Adenocarcinoma↗
PubMed1996

Nuclear morphometry in squamous cell carcinoma (SCC) of the tongue.

In the histological grading of oral squamous cell carcinoma (OSCC) nuclear features are very important. Nevertheless evaluation is usually performed in a subjective and not highly reproducible way. The aim of this work was to investigate the relationship between nuclear shape and survival in 30 cases of carcinoma of the tongue. All the patients were divided into two groups: short-term survival and long-term survival. Twenty nuclei for each tumour were submitted to a morphometrical study by the shape analytical morphometry (SAM) software system. It was thus possible to evaluate not only nuclear dimensions but also nuclear contour irregularities and nuclear shape asymmetries. Multivariate discriminant analysis (MDA) of the quantitative parameters obtained by the morphometrical study distinguished the patients of the two groups with only a 10% error; moreover successful cluster analysis was performed by using Fourier parameters. Both these sets of results were achieved mainly owing to the parameters for contour irregularities.

Adult↗
PubMed1996

Nuclear morphometry in node-negative breast carcinoma.

OBJECTIVE: To determine whether nuclear morphometry can confirm or add useful information to classic clinicopathological prognosticators to identify the subpopulation of breast carcinoma patients with node-negative (N-) disease, at high risk of disease relapse. STUDY DESIGN: On the basis of results obtained by clinicopathologic evaluation of a group of patients with N- breast cancer, on a test group of 56 cases (32 patients disease free and 24 with relapse), we performed a morphometric analytical study of nuclei using the Shape Analytical Morphometry (SAM) software system; 20 nuclei for each case and 17 morphometric parameters for each nucleus were analyzed. RESULTS: The SAM system allowed us to quantify shape differences in nuclei in terms of contour irregularities and asymmetries along with evaluation of nuclear dimensions. Dimensional and analytic parameters were subjected to univariate (Student's t test) and multivariate (Hotelling's test) analysis. Multivariate discriminant analysis showed that an exact forecast of disease relapse could be made in 77% of patients with N- breast cancer by using a set of six both analytic and dimensional parameters. CONCLUSION: These results confirm that nuclear pleomorphism is the result of both contour irregularities and shape asymmetries and that even though they should be considered preliminary results, they stress the importance of quantitative shape evaluation.

Breast Neoplasms↗