Post-transcriptional regulation of HSP70 expression following oxidative stress in SH-SY5Y cells: the potential involvement of the RNA-binding protein HuR.

Brain aging is associated with a progressive imbalance between intracellular concentration of Reactive Oxygen Species (ROS) and cells ability to activate defensive genes. Heat Shock Protein 70 (HSP70) has been shown to act as a fundamental defensive mechanism for neurons exposed to an oxidant challenge, and its expression decreases during senescence. In the present report we show that the RNA-binding protein ELAV/HuR can affect, post-transcriptionally, the fate of HSP70 mRNA following H(2)O(2)-mediated oxidative stress in SH-SY5Y human neuroblastoma cells. As a consequence of H(2)O(2) treatment (1mM for 30 minutes), HSP70 mRNA accumulates in the ribosomes associated to the cytoskeleton, where parallel Western blotting experiments reveal statistically significant increase for both HuR and HSP70 protein levels. We also confirm the capability of HuR to bind to HSP70 mRNA, and describe how the biological effect of this ELAV protein on the HSP70 mRNA could be due to a direct phosphorylation in serine/threonine residues of HuR itself by the early (10 minutes) H(2)O(2)-mediated activation of PKC alpha. Our findings shed light on the post-transcriptional regulation of HSP70 expression, suggesting the existence of a new molecular cascade -involving PKC/HuR/HSP70- that possibly represents an early event in the cellular response to H(2)O(2)-mediated oxidative stress in SH-SY5Y human neuroblastoma cells. The present results lead us to speculate that an impairment in this regulatory mechanism might directly contribute to the defective cellular response to oxidative stress, thus helping to dissect a potential tool useful to counteract some aspects associated to cerebral senescence.

Characterization of two novel cell lines, DERL-2 (CD56+/CD3+/Tcry5+) and DERL-7 (CD56+/CD3-/TCRgammadelta-), derived from a single patient with CD56+ non-Hodgkin's lymphoma.

Two novel IL2-dependent cell lines, DERL-2 and DERL-7, were established from a patient with hepatosplenic gammadelta T cell lymphoma. This patient presented, at diagnosis, two discrete populations of CD56+ cells, one TCRgammadelta+, the second lacking T cell-restricted antigens. The cell lines derived displayed features corresponding to the two cellular components of the disease: DERL-2 was CD56+/CD3+/TcRgammadelta+ while DERL-7 was CD56+/CD3-/TcRgammadelta-. Along with CD56, the two cell lines shared the expression of CD7, CD2, CD158b and CD117. Karyotype analysis showed that both cell lines were near-diploid, with iso-7q and loss of one chromosome 10. In addition, DERL-2 showed 5q+ in all metaphases analyzed, while DERL-7 revealed loss of one chromosome 4. Genotypically, both cell lines shared the same STR pattern at nine loci and demonstrated an identical rearranged pattern of the T cell receptor genes beta, gamma and delta, with respect to the original tumor cells. These data indicated that both cell lines and the original neoplastic populations were T cell-derived and arose from a common ancestor. Among a large panel of cytokines tested, only SCF was able to substitute IL2 in supporting cell proliferation. Moreover, SCF and IL2 acted synergistically, dramatically enhancing cell growth. These cell lines may represent a model to further analyze the overlap area between T and NK cell malignancies, and may provide new information about the synergistic action of IL2 and SCF on normal and neoplastic T/NK cells.

Multivariate discriminant function based on six biochemical markers in blood can predict the cirrhotic evolution of chronic hepatitis.

BACKGROUND: Serologic markers have been proposed for monitoring hepatic fibrosis in chronic active liver disease. Because none of these markers, when used singly, is totally satisfactory, we developed and evaluated a multivariate approach. METHODS: We studied two cohorts of chronic hepatitis (54 patients) and cirrhosis patients (49 patients) to identify a panel of biochemical markers that discriminates between the two diseases. Using multivariate discriminant analysis, we selected a function, based on the concentrations of six biochemical markers (fibronectin, prothrombin, pseudocholinesterase, alanine aminotransferase, manganese superoxide dismutase, and N-acetyl-beta-glucosaminidase). We then prospectively validated this function on a second temporal cohort of patients. RESULTS: Multivariate discriminant analysis correctly classified 93.7% of patients (94.3% of chronic hepatitis and 92.9% of cirrhosis patients) in the first cohort and 85% of patients (89.5% of chronic hepatitis patients and 81% of cirrhosis patients) in the second cohort. CONCLUSIONS: Discriminant analysis of results of six inexpensive biochemical markers provides a high predictive value for differentiation between liver cirrhosis and chronic hepatitis. Consequently, these biochemical markers condensed into a multivariate discriminant analysis value for each patient provide information that can be contributory for subsequent options during the evolution of the natural history of chronic hepatitis.

The horizontal transfer of Agrobacterium rhizogenes genes and the evolution of the genus Nicotiana.

With the aim of understanding better the distribution and evolution of Agrobacterium rhizogenes genes transferred in the genus Nicotiana, 42 species were screened for presence of rolB, rolC, ORF13, and ORF14. The transferred sequences were then compared within the genus and with current bacterial sequences. The results obtained showed the presence of at least one bacterial gene in 15 species belonging to different subgenera. Sequence analyses supported the hypothesis of coevolution of bacterial and plant sequences, thus suggesting a possible role for the transferred genes in the early events of Nicotiana species differentiation. The high level of conservation of Agrobacterium sequences and the dependence of their expression from the plant physiological context along with previous data suggesting their involvement in the determination of the plant hormonal balance were all consistent with this hypothesis. The results are finally discussed also as to their relevance for the hypothesis of mono and multi ancient infection by Agrobacterium.

Effect of N-acetyl-cysteine on lymphomonocyte glutathione and response to interferon treatment in C-virus chronic hepatitis.

BACKGROUND/AIM: Much controversy exists concerning effect of N-acetyl-cysteine, a precursor of glutathione, on the response to interferon treatment in patients with C-virus chronic hepatitis. The aim of this study was to evaluate the efficacy of interferon therapy with and without oral N-acetyl-cysteine. We also measured glutathione concentrations in lymphomonocytes of 25 patients with chronic C-virus hepatitis before and after interferon treatment and correlated the results with treatment response. METHODS: Glutathione was extracted from lymphomonocytes and measured with a modified high performance liquid chromatographic method in the 25 hepatitis patients and 12 healthy controls. RESULTS/CONCLUSIONS: 1) Hepatitis patients and controls had similar basal concentrations of lymphomonocytic glutathione; 2) neither interferon nor N-acetyl-cysteine significantly affected glutathione concentrations in patients; and 3) N-acetyl-cysteine did not affect response to interferon.

A physiological and molecular analysis of the genus Nicotiana.

An analysis of the evolution of the genus Nicotiana was carried out with physiological and molecular tools. The capacity of explants from seedlings of several species of Nicotiana to differentiate roots or shoots or to habituate was used to ascertain whether the in vitro behavior of species has a nonrandom distribution in the genus. The results obtained allowed us to identify two groups of species, one root-forming prone composed of Paniculatae (subgenus Rustica) and the other composed of Alatae, Repandae, and Noctiflorae (subgenus Petunioides), with a major tendency toward the production of shoots. Habituation capacity was characteristic of species randomly distributed throughout the phylogenetic tree. These data suggest fixation throughout the evolution of coadapted gene complexes (hormone-related genes) involved in the control of developmental processes. RAPDs, on the other hand, used as molecular markers for the clustering of related species, seem entirely coherent both with classical morphological and karyological studies and with in vitro physiological methods, supporting an early subdivision of the whole genus into two diverging developmental patterns.

Ascitic pseudouridine discriminates between hepatocarcinoma-derived ascites and cirrhotic ascites.

Various biochemical indexes discriminate neoplastic from nonneoplastic ascites. However, within the latter group, the distinction between cirrhotic ascites and ascites caused by hepatocarcinoma (HC) is usually based on liver biopsy or cytology. HC-derived ascites is included in the group of nonneoplastic ascites because it is not associated with peritoneal spreading of neoplastic cells. In 54 cases of cirrhotic ascites and 17 cases of HC ascites, all histologically diagnosed, ascitic pseudouridine concentrations discriminated cirrhotic from HC ascites. For example, using the cutoff value of 4.25 mumol/L (obtained by ROC curve analysis) resulted in a diagnostic sensitivity of 88.2% and a diagnostic specificity of 90.8%. Moreover, in cirrhosis, the ascitic concentrations of pseudouridine were lower than serum concentrations, and the two sets of values were correlated; in HC, however, ascitic pseudouridine concentrations were higher than serum concentrations, and the two were unrelated. These findings strongly suggest that in cirrhotic patients ascitic pseudouridine derives from serum by diffusion, whereas in HC patients the mechanism appears to be more complex.

Pseudouridine and 1-ribosylpyridin-4-one-3-carboxamide (PCNR) serum concentrations in human immunodeficiency virus type 1-infected patients are independent predictors for AIDS progression.

A prospective study was conducted with 161 human immunodeficiency virus (HIV)-positive patients to investigate the prognostic role of 10 serum-modified nucleosides with regard to some of the most widely used parameters of AIDS progression. Serum concentrations of pseudouridine (> 3.77 nmol/mL) predicted progression to AIDS in CDC stage A2 HIV-infected patients much better than did other widely used parameters (hazard ratio, 2.7; 95% confidence interval, 1.29-6.35; P = .01; median permanence time in stage A2, 17 vs. 30.5 months; P = .03). Serum concentrations of 1-ribosylpyridin-4-one-3-carboxamide (PCNR) and beta 2-microglobulin and the CD4:CD8 cell ratio, in decreasing order and used in combination, differentiated the overall survival time probability of AIDS patients; PCNR was the best and a new independent predictor (overall survival time, > 31 months, no positive parameters; 19.3 months, one positive parameter; and 5.5 months, two positive parameters.